Serum Leukotriene Metabolism and Type I Hypersensitivity Reactions in Different Animal Species

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Graduate Student Theses, Dissertations, & Professional Papers Graduate School

1989

Serum leukotriene metabolism and Type I hypersensitivity reactions in different animal species

Thulasi Sarojam Unnitan The University of Montana

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SERUM LEUKOTRIENE METABOLISM AND TYPE I HYPERSENSITIVITY

REACTIONS IN DIFFERENT ANIMAL SPECIES

By Thulasi Sarojam Unnithan M.B.B.S., Trivandrum Medical College, India, 1981

Presented in partial fulfillment of the requirements

for the degree of Master of Science

University of Montana,

1989

8^'an, Graduate School

($hL& . & Date UMI Number: EP34626

INFORMATION TO ALL USERS The quality of this reproduction is dependent on the quality of the copy submitted.

In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.

Dissertation Publishing

ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Unnithan, Thulasi S. M.S., December 198 9 Microbiology Serum Leukotriene Metabolism and Type I Hypersensitivity Reactions in Different Animal

Director: Dr. George L. Card

The peptidoleukotrienes, leukotriene C4, leukotriene D4 and leukotriene E4/ (LTC4, LTD4, and LTE4) which are products of the 5-lipoxygenase pathway of arachidonic acid metabolism, play important roles in mediating hypersensitivity reactions. LTC4 is the primary leukotriene released from stimulated cells. A transpeptidase activity converts LTC4 to LTD4, which is subsequently converted to LTE4 bya dipeptidase activity. LTC4 metabolism was studied in human, mouse, guineapig, rabbit, horse and cow sera. LTD4 is the most potent of the peptidoleukotrienes. The relative amounts of LTD4 present after specific periods of incubation was compared in the sera of different species, 3 using tritium labelled LTC4 ( H-LTC4) After specific incubation time, the lipids were extracted from the serum, and the leukotrienes separated by Reverse Phase High Pressure Liquid Chromatography. The leukotrienes in the different fractions were quantitated based on the radioactivity in the corresponding fractions. The profile of metabolism was different for each species in that the rate of formation of LTD4 (transpeptidase activity) and the rate of LTD4 metabolism (dipeptidase activity) varied in the different species. On the basis of these two enzymatic activities, the different animal species tested could be divided into four categories; (1)those with high transpeptidase and dipeptidase activity, which would result in large amount of LTD4 accumulation initially, with a rapid decline, as in horse and cow:(2)those with low activity of transpeptidase and dipeptidase, resulting in accumulation of formed LTD4, as in guineapig;(3)those with high transpeptidase activity and a low dipeptidase activity which would result in accumulation of large amounts of LTD4, as in humans;(4)those with low transpeptidase and high dipeptidase activitywhich results in very low or no accumulation of LTD4 as in mouse. The results show a relationship between the relative amounts of LTD4 observed and the manifestation of Type I hypersensitivity in the different species. ACKNOWLEDGEMENT I wish to express my gratitude to my mentor and major professor, Dr. George L. Card, for his constant guidance and encouragement. I also wish to thank Dr. Barbara E. Wright, Dr. David Freiman and Dr. Dan C. DeBorde for their timely help and advice. I thank my husband, Mr. Sureshkumar C. Nair, for his constant support and encouragement, without which this would have been impossible. A special word of thanks to Mr. Michael J. Rhodes, whose help throughout the course of this work is very much appreciated; and to Karyn Taylor, Dr. Lee Yi-Ping and other friends who have helped in so many ways during my stay in Missoula.

iii TABLE OF CONTENTS

Abstract ii

Acknowledgement iii

Table of contents iv List of figures v

List of tables vii Introduction 1

Materials and Methods 20 Results 27 Discussion 60 References 72

iv LIST OF FIGURES: Fi no. Page no.

1. Mechanisms involved in release of arachidonic acid.. 2

2. The arachidonic acidcascade 4 3. Metabolism of the peptidoleukotrienes 7 4. Chromatogram of standard leukotrienes 28 5. Buffer control, and RBL-1 metabolized leukotriene C4 29

6. Chromatogram of LTC4 in buffer, serum at 40C, and

protein denatured serum 32 7. Chromatogram of LTD4 in buffer, serum at 40C and

protein denatured serum 33

8. 3H-LTC4 metabolism in human serum 34 9. Difference in 3H-LTC4 metabolism between clotted and heparinized samples 35

10 Difference in 3H-LTC4 metabolism between stored and

fresh samples 36

11 Relationship between volume of serum and 3H-LTC4

conversion 37

12 Time course using 3H-LTC4 in human serum 38

13 Substrate conc. vs. percent conversion of 3H-LTC4... 39

14 Profile of3H-LTC4 metabolism in human and mouseserum.42

15 3H-LTC4 metabolism in human and mouse serum-time course

study 43

16 Time course for 3H-LTD4 in human and mouse sera.... 44

17 Substrate covcentration vs. 3H-LTD4 conversion 45

v 18. Volume (dilutions) vs. 3H-LTD4 conversion 46 19. Dipeptidase activity in normal vs. heat inactivated human and mouse sera 47 20. Dipeptidase activity in normal human in serum the presence of heat inactivated human serum 48

21. Effect of heat inactivated mouse and human serum on

dipeptidase activity in normal mouse and humanserum. 4 9

22. Time course for 3H-LTC4 conversion - Human, mouse,

guinea pig and rabbit sera 51 23. Time course for 3H-LTD4 conversion -Human,mouse, rabbit and guinea pig sera 52 24. Relative amounts of LTD4 and LTE4 present after 20

and 90 minute incubation 53

25. Relative amounts of LTD4 present, as percent of total 3H-LTC4 converted in human, mouse, rabbit and guinea pig sera 54

26. LTC4 conversion in human, mouse, rabbbit, guinea pig, horse, cow and Fetal bovine sera 55

27. LTD4 metabolism in human, mouse, rabbit, guinea pig,

horse, cow and Feyal Bovine sera 56

28. Relative amount of LTD4 presnt in human, mouse, rabbit,

guinea pig, horse,and cow serum after 15, 60, and 90 minutes 57

29. Relative amounts of the unknown radioactive substance

present, compareed to 3H-LTD4 metabolism 58

vi LIST OF TABLES Page no.

Table I. Mean and S.D. values of percent conversion of LTC4 and LTD4 in six serum samples 31

Table II. Values obtained for duplicate samples for LTC4 and LTD4 conversion insera of different animal species 59

vii INTRODUCTION

Arachidonic acid (a 20-C polyunsaturated fatty acid), is a 5,8,11/14-eicosatetraenoic acid. Arachidonic acid (AA) and its metabolites are known to play important roles in mediating hypersensitivity reactions like bronchial asthma, allergic rhinitis and psoriasis (24, 32) .

Arachidonic acid is usually present in membrane phospholipids (phosphatidyl choline, phosphatidyl inositol, etc.) esterified primarily to the second carbon (C-2) position. Release of AA from the C-2 position is mediated by different types of stimuli, which can be physiological or pathological (64). Hormones like angiotensin, bradykinin, epinephrine, antigen-antibody complexes etc. are physiological stimuli, whereas ischaemia, mechanical damage, membrane-active venoms (like mellitin from bee venom), Ca2+ ionphores (A23187), phorbol esters etc. are pathological stimuli. The membrane AA is released by the effect of two phospholipase: phospholipase C, which is specific for phosphatidyl inositol, and phospholipase A2, which is specific for phosphatidyl choline (Fig. 1). In general, all stimuli lead to changes in cell membrane, which lead to transport of ions across the membrane, especially a flow of

Ca2+ ions from the extracellular to the intracellular

1 2

PI-SPECIFIC PHOSPHOLIPASE C (INACTIVE)

PHOSPHATIDYL Co4"4 INOSITOL MOBILIZATION

PI-SPECIFIC PHOSPHOLIPASE C (ACTIVE)

DIGLYCERIDE

PROTEIN GLYCERIDE KINASE C LIPASE (s ) (INACTIVE) (+) MONOGLYCERIDE

ARACHIDONIC PROTEIN ACID KINASE C (ACTIVE) PHOSPHOLIPASE A PHOSPHO-INHIBITORY PROTEIN INHIBITORY PROTEIN- + COMPLEX PHOSPHOLIPASE A2 ADP

PHOSPHATIDYL l-ACYL-GPC + CHOLINE ARACHIDONIC ACID

Fig. 1. Mechanisms involved in release of arachidonic acid. 3 compartment. The resulting increased intracellular Ca2+ concentration stimulate the cytoplasmic phospholipase C, which acts on phosphatidyl inositol to give

inositoltriphosphate (IP3) and diacylglyceride (DAG). DAG

is acted upon by other lipases to a monoacylglyceride and

AA. IP3 induces further Ca2+ mobilization from the endoplasmic reticulum to the cytoplasm, and this further activates phospholipase C. The DAG which is not converted to monoglyceride, stimulates other enzymes. Protein kinase

C (PKC) is a major enzyme activated by DAG. PKC

phosphorylates a phospholipase A2 inhibitory protein. This causes activation of phospholipase A2, which cleaves AA from phosphatidyl choline (64). The released AA has a very short half life. It is either metabolized, by a cyclooxygenase or

a lipoxygenase pathway, or is reincorporated into the

membrane phospholipids. Very small amounts of AA are found

in the free fatty acid pool. This low level is thought to

be maintained by the action of an acyl-CoA transferase, which esterifies arachidonyl-CoA into phospholipids (25). The metabolism of AA by the lipoxygenase and the cyclooxygenase pathways are together known as the arachidonic acid cascade (Fig. 2). Metabolism by the cyclooxygenase pathway leads to the formation of prostaglandins (PGs). The first PG to be formed is PG-G2.

This is converted to PG-H2 from which other PGs and thromboxanes are formed. 4

phospholipids

Arachidonic acid

15-Lipoxygenase cyclooxygenase 5-lipoxygenase 12-lipoxygenase)

12-HPETE PG-G; 5-HPETE 15-HETE iipoxins 5-HETE 12-HETE 14,15diHETE

1TA, TxB

PGF; PGD2 PGE2 ?GI LTB, •ans.diastereo LTD. sulfoxide 2Q-0H LTS, metab' 2Q-COOH L'

Fig. 2. The arachidonic acid cascade. 5

The lipoxygenase pathways involve metabolism by 5-, 15- and 12-lipoxygenases. Lipoxins are produced by the 15- lipoxygenase pathway, and hydroxy eicosatetraenoic acids (HETES) are produced by the 12-lipoxygenase pathway.

The 5-lipoxygenase pathway converts AA to 5- hydroperoxy,6,8,11,14 eicosatetraenoic acid (5-HPETE). 5- HPETE can then undergo enzymatic conversion to 5-HETE or to

4 5,15,diHETE. Leukotriene A (LTA4) is formed from 5-HPETE by an LTA4 synthase. LTA4 is very unstable. The action of an LTA4 hydrolase converts LTA4 to LTB4, which is relatively stable. LTB4 can also be formed from 5-HETE, in the presence of cytochrome P-450 (64). Conversion of LTB4 to its wOJ -metabolites by Polymorphonuclear cells (PMNs) has been reported. 20-carboxy LTB4 and 20-hydroxy LTB4 are the products of Co -oxidation.

LTA4 undergoes enzymatic conversion to LTC4 by the action of a glutathione-S-transferase, which is specific for

LTA4. Addition of a glutathione residue to the C-6 position by a sulfo-ether linkage, leads to the formation of LTC4, which is the first peptidoleukotriene to be formed. LTC4, along with its metabolites, LTD4 and LTE4 were formerly known as the Slow Reacting Substances of Anaphylaxis

(SRS-A), Although SRS-A has been identified as early as

1930 it was only in 1980 that it was found to be a mixture of peptidoleukotriens (7). The main component of the SRS-A is LTD4 (48). The name leukotriene refers to the 6 observation that they were seen to be released by leukocytes, and had three conjugated double bonds.

LTC4 is the form in which the peptidoleukotrienes are

released into the extracellular medium. LTC4 is converted to LTD4 by transpeptidase reaction (64) that removes the

glutamate from the glutathione residue. LTD4 is further

converted to LTE4 by a dipeptidase activity (64), which

cleaves the glycine from LTD4. Several cell types are involved in the release of

leukotrienes. LTB4 is released primarily from the PMNs (57,65). It is also released from bronchi and lung

parenchyma (68). LTC4, on the other hand, is released predominantly from the eosinophils (57,65), mast cells,

alveolar macrophages (24),and in lung, parenchymal tissue (68).

The metabolism of the peptidoleukotrienes has been studied in detail, both in vivo, and in vitro. In-vitro

studies, showed conversion of LTC4 to LTD4 in Rat Basophil

Leukemia (RBL-1) cells (42). The conversion of LTD4 to LTE4 was also first detected in RBL-1 cells (43). In fact, the

SRS-A was first shown to be a metabolite of AA in RBL-1 cells (27). The metabolism of leukotrienes has also been studied in human plasma (1,31), and in the supernatant of

PMNs (51). The transpeptidase activity responsible for the

formation of LTD4 is released by PMNs, as seen by the formation of LTD4 from LTC4 in the supernatant of a 7

COOH

"S-CH

CHCONHCHjCOOH NHCOFCH^CHCOOH NHj LTC, V-GTP

COOH

S- CH

CHCONHCHjCOOH

COOH

S-CH

CHCOOH

coo- MNCOC *- £>. ^COCH,

20-COOH LTt,*N Ac ?(VOH LTl -NA;

Fig. 3. Metabolism of the peptidoleukotrienes. 8 suspension of PMNs (51). The transpeptidase activity and dipeptidase activity were seen to be released from PMNs following immunological as well as nonimmunological stimuli (54). The transpeptidase activity was seen to be localized

in the microsomal fraction of PMNs, by subcellular

fractionation (51). This activity has also been detected in plasma (1), in the supernatant of alveolar macrophages,

(15), and in the parenchyma of human and guinea pig lung tissue (53,54). The transpeptidase activity that converts

LTC4 to LTD4 is thought to be due to a Y*-glutamyl transferase, as glutamate was seen to be a substrate for this enzyme (23) and since inhibitors of Y-glutamyl transferase (L-serine borate, avicin) (43) inhibited the

formation of LTD4.

Other than conversion to LTD4, LTC4 can also undergo oxidative metabolism to biologically inactive products (33). This reaction was seen to take place during the respiratory burst in PMNs. Sulfoxides of LTC4, like LTC4 chlorsulfonium

ion, are metabolites of this oxidative metabolism.

LTD4 is converted to LTE4 by a dipeptidase activity (64), which was associated with specific granules of PMNs

(34 ), and plasma membrane of RBL-1 cells (51). This activity is present in alveolar macrophages, and in lung parenchyma, as shown by the presence of LTE4 in these tissues following antigen challenge (17). L-cysteine is a dipeptidase inhibitor (43). The dipeptidase activity also 9 requires the presence of divalent cations. Substances like L-cysteine and dithiothreotol, which bind divalent cations, decreased the dipeptidase activity, which was subsequently restored by the addition of Co2+, Zn2+, or Mn2+ into the system (52).

LTE4 is relatively stable. It can undergo further conversion to N-acetyl LTE4 in the presence of acetyl Co-A.

14 This has been shown by the use of C-Acetyl CO-A (23) . LTE4 and N-acetyl LTE4 are the main excretory metabolites of peptidoleukotriene metabolism in man and other mammalian species (6,60). N-acetyl LTE4 can also be converted to 20- carboxy N-acetyl LTE4 and 20-hydroxy N-acetyl LTE4 (23) .

Addition of a glutamate residue to LTE4 by a Y-glutamyl transferase leads to the formation of LTF4, which is a V- glutamyl-cysteinyl leukotriene(23).

Other than arachidonic acid, trienoic and pentaenoic acids were also seen to be metabolized to peptidoleukotrienes LTC3 and LTCS, as seen by the presence of LTE3 and LTES in RBL-1 cells. These leukotrienes, with 3 or 5 double bonds, were seen to cross react with anti-LTC4 antibodies. The biological activity and enzyme specificities of these leukotrienes were seen to be similar to that of the leukotrienes with four double bonds (43).

The biological activities of the different leukotrienes have been described (55) . LTB4 is mainly chemotactic in activity. It facilitates migration of all leukocytes, but 10 the PMNs are mainly affected. LTB4 also increases microvascular permeability. The peptidoleukotrienes, also referred to as the cysteinyl leukotrienes, have a smooth muscle constrictor effect, and thus induce bronchospasm.

LTC4, LTD4 and LTE4 are reported to be 100 to 1, 000 times as potent as histamine, on a molar basis (48,55) . Other than their direct constrictor effect on airway smooth muscle, they can also potentiate the action of other inflammatory

mediators. LTC4 and LTD4 increase microvascular permeability and mucous secretion in bronchioles. These activities suggest that the cysteinyl leukotrienes are the major mediators of type I hypersensitivity reactions like bronchial asthma, allergic rhinitis and anaphylactic shock. Four types of hypersensitivity reactions are recognized: Type I, Type II, Type III and Type IV (64). These are inflammatory reactions, mediated by the release of pharmacologically active substances like histamine and leukotrienes, from specific cell types, following antigen- antibody reactions. The Type I reactions are more acute, and cause more discomfort. This type of reaction is referred to as immediate hypersensitivity reaction, allergy or anaphylaxis. Antigen-antibody reactions cause release of AA and its metabolites directly from cell membranes of mast cells. AA metabolites, like leukotrienes, which are mediators of hypersensitivity reactions are also released directly by eosinophils, which are attracted to the site of 11 the reaction by chemotactic substances released by mast cells. These substances are called eosinophil chemotactic substances of anaphylaxis (ECF-A). The symptoms of Type I

hypersensitivity can be attributed to the sum total of all

the above factors. Bronchial asthma, which is a Type I

hypersensitivity reaction , is characterized by severe prolonged bronchoconstriction and airway obstruction, both of which are due to the release of these mediators. In

fact, several workers have reported the presence of elevated

plasma levels of cysteinyl leukotrienes during the asthmatic attack (40,56,68). Eosinophils from asthmatic individuals have been reported to produce larger amounts of

LTC4 than those from normal individuals (59). The Type II hypersensitivity reaction results in cell destruction due to antigen-antibody reactions. Blood group incompatibilities fall into this group. The Arthus reaction, acute and

chronic serum sickness which come under the Type III

hypersensitivity reaction are produced by localized tissue

damage due to deposition of immune complexes. Type IV

hypersensitivity reactions are cell mediated reactions, in response to localized injection of antigen. It is mediated through lymphocytes and is called delayed hypersensitivity reaction (DHR).

The metabolism of LTB4 in human blood has been studied (71). It was seen that LTB4 slowly undergoes co- oxidation, to 20-hydroxy and 20-carboxy LTB4. There was no difference in the rate of metabolism between normal and asthmatic individuals, suggesting that this pathway may not

influence the outcome in a hypersensitivity state. The cysteinyl or peptidoleukotrienes have been

implicated in a wide variety of hypersensitivity disorders

eg., bronchial asthma (56,59), psoriasis (10) and allergic rhinitis (67). In an allergic condition, the eosinophils, which release the peptidoleukotrienes, are thought to be

attracted to the tissue by the LTB4 released by the PMNs

(57). LTC4 is the form in which the SRS-A is released from stimulated cells. Therefore, the high levels of LTD4 detected in the plasma of individuals during the asthmatic

attack (40), and the LTD4 and LTE4 seen following antigen challenge in human (17) and guinea pig (53) lung, must be due to the presence of metabolizing enzymes released into the medium. The release of these enzymes following antigen challenge have been shown by several workers (15). An elevated amount of leukotriene release has been reported from the lung (14), and stimulated blood cells (eosinophils) of individuals with allergic asthma and chronic bronchial asthma (59). This observation suggests that an increased release of leukotrienes might be the factor that predisposes these individuals to the development of hypersensitivty reactions.

A comparison of the urinary LTE4 levels in normal and atopic individuals after antigen challenge, and in asthmatic 13 individuals during the asthmatic attack, showed that although the LTE4 levels were elevated in atopic individuals following antigen challenge, they were not elevated in individuals during the acute asthmatic attack, or in individuals having a severe episode of allergic rhinitis (60). These results suggest that if an increased release of LTs does occur during an acute asthmatic attack, there might be an alteration in the further metabolism of these leukotrienes to a biologically less active and excretable form, as increased levels were not detected in the urine during the acute attack. Bronchial asthma is an anaphylactic reaction. Therefore , anaphylaxis induced in animal models should serve as an excellent model to study the role of leukotriene metabolism in bronchial asthma, and in hypersensitivity reactions as a whole. Although the phenomenon of hypersensitivity was observed in 1839 by Magendie, in rabbits, it was Richet, in 1902, who first recognized the reaction in dogs and named it "anaphylaxis" meaning "without protection" (50). Theobald Smith, in 1905, noted the ease with which guinea pigs developed anaphylaxis as compared to other animals (50). Thus guinea pigs became the experimental animal of choice to elicit the anaphylactic reaction in future experiments. The symptoms of anaphylaxis in guinea pigs were seen to resemble those in humans. The bronchoconstriction observed, was similar to that in 14 bronchial asthma (Burson, 1911, as quoted by Alexander, 1926) (3). Later, Chase reported that it was possible to elicit the cutaneous anaphylactic reaction in guinea pigs, just as in humans. Experiments conducted by Rabat in 1942 (28), indicated that very small amounts of antigen was

sufficient to induce anaphylaxis in guinea pigs. Isolated guinea pig smooth muscle was also found to have the same

amount of responsiveness to antigen as the intact animal. The amount of antigen required to produce fatal anaphylaxis was also found to be very small (29). Guinea pig smooth muscle was used as early as 1926 to elicit the Schultz-Dale

reaction, in order to elicit the sensitivity of smooth muscle to antigen. This experiment is used widely, to study the effect of several mediators of anaphylaxis, because the

symptoms of anaphylaxis are due to smooth muscle

constriction. In many recent reports, the guinea pig has been used to elicit the anaphylactic reaction, in order to study the role of leukotrienes in anaphylaxis (30,53,62,63).

Release of leukotrienes from trachea (53) and lung (62) of guinea pig following antigen challenge has been reported.

These findings are also similar to reports of identical experiments conducted on human lung tissue (54).

The mechanism of anaphylaxis was studied in mice by Weisser and coworkers in 1941 (69). A series of experiments were conducted to determine the susceptibility of mice to develop anaphylaxis after active and passive sensitization. 15 The mice responded with little or no signs of anaphylaxis, and none showed fatal anaphylaxis. Hours after unilateral adrenalectomy, two mice had fatal shock, while the majority of animals died of shock after bilateral adrenalectomy. Sobey and Adams, in 1959, used a score, called anaphylactic score, to study the anaphylactic reaction in a batch of mother and daughter mice (58). This was based on symptoms like itching and decreased activity. A wide variation was found in the anaphylactic score in different mice. Based on these observations, they concluded that the anaphylactic potential of a mouse was determined by that of its mother and that this potential was under genetic control. The development of passive cutaneous anaphylaxis (PCA) was also found to be much slower in mice than in guinea pigs, and required a much larger dose of antigen (45). These findings were later supported by Levy in 1964 (35), who developed a technique to elicit PCA and reverse passive Arthus reactions simultaneously. A difference was noticed between three animal species. The mouse was found to be least sensitive to these cutaneous reactions, showing only a mild reaction to undiluted antigen. The rat was about 10 times more sensitive than the mouse, responding to 1/10 dilution, whereas the guinea pig was 1,000 times more sensitive than the mouse, since anaphylaxis was induced by a 1/1,000 dilution of the antigen. The difficulty in eliciting anaphylaxis and fatal shock in rats has also been described 16 (49). Bilateral adrenalectomy was necessary to induce signs

of fatal shock. The difficulty in inducing the Arthus phenomenon in rats, as compared to guinea pigs and rabbits

has also been described (41).

The development of anaphylaxis in rabbits has been described as early as 1911 (Auer, as quoted by Coca in 1919) (16). The anaphylactic reaction in rabbits does not

resemble that in humans. Although during anaphylaxis symptoms of respiratory distress were present, bronchoconstriction was not seen in rabbits. The cause of death in fatal shock was cardiac failure, due to increased

pulmonary arterial pressure, as opposed to bronchiolar spasm in guinea pigs (50). This is explained by the fact that in rabbit, the pulmonary artery has a well developed smooth muscle layer, while the bronchiolar musculature does not. Manwaring used rabbit smooth muscle to study the response to

antigens (36). It was found that although the animal developed fatal shock, smooth muscle contraction, as recorded by rise in intra cystic pressure, was very rare in rabbits, when compared to guinea pigs. Recent experiments have shown that LTC4 and LTD4 were seen to produce strong contraction in guinea pig lung parenchyma, while in rat and rabbit lung parenchyma, the dose required to produce a similar contraction was 3-4 fold higher (47).

Horse and cattle anaphylaxis are noted to have some similarity to human reactions (50). Dyspnoea, increased 17 peristalsis, and sweating are the main symptoms. In horses, the anaphylactic response is characterized by acute hypotension, followed by increased pulmonary arterial pressure and respiratory distress within 8-12 minutes. In cattle, similar symptoms are described, with severe reactions taking 15-20 minutes to manifest themselves. In both species death is reported to occur in 2-5 minutes (8). In the calf, abdominal distension is a marked feature. This was explained by the presence of a strong smooth muscle layer in the stomach compartments (50).

It is clear from the above data that a difference exists in the susceptibility of different species to develop anaphylaxis. The role of the peptidoleukotrienes in anaphylaxis has also been established. Studies have been done on in-vivo LTC metabolism in different species and differences in the metabolite that accumulates after specific periods of time have been reported (4, 23,44). In vitro studies have been done to study the products of metabolism and the comparative uptake of LTC by isolated organs and cells (23). The results of these studies showed a differential uptake by different organs. Metabolism of

LTC4 has been studied in human plasma. Although human plasma was seen to convert LTC4 to LTD4 and LTE4 (1,31), LTD was not seen in the mouse or guinea pig tissues in in-vivo studies using LTC (4,22) . In vivo studies on the metabolism of leukotrienes in different species has also shown that the 18 form in which the leukotrienes are excreted differed with the species. In humans, LTE4 was the main excretory metabolite of LTC4 metabolism (60,66), while in monkeys, N- acetyl LTE4 was the form in which the peptidoleukotrienes were excreted (6). In rats, N-acetyl LTE4 was the main metabolite (44). Considering these observations, it seemed likely that the profile of LTC4 metabolism in the serum of these animal species would differ. This assumption was supported by observations made during our preliminary experiments using human and mouse serum (11). The profile of LTC4 metabolism in mouse serum was seen to be very different from that in human serum, in that, after a specific incubation period, no LTD4 was detected in mouse serum; most of the leukotriene detected was in the form of

LTC4, with small amounts present in the LTE4 fraction. Under the same conditions, human serum showed presence of significant amounts of LTD4 and LTE4, with very little LTC4.

Since LTD4 is known to be the most potent biologically among the peptidoleukotrienes, this observation correlated with the present knowledge concerning the susceptibility to the development of anaphylaxis in mice and humans.

This study is intended to compare the profiles in LTC4 and LTD4 metabolism in human, mouse, rabbit, guinea pig, horse, cow and fetal calf sera. THESIS OBJECTIVES: The role of peptidoleukotrienes in anaphylaxis has been established. A species specific susceptibility to develop anaphylaxis has also been described. The main objectives of this study are to determine 1) if a species specific difference exists in the metabolic profile of LTC4, which is the form in which the peptidoleukotrienes are released, and 2) if the anaphylactic susceptibility of an animal correlates with the profile of its LTC4 metabolism. MATERIALS AND METHODS

Collection of Animal Blood and Sera Human blood collected from normal human volunteers, with no history of asthma or other allergic manifestations, was drawn from the cubital vein into heparin free vacutainers. The source of mouse blood was Balb-C mice in all experiments except the last one, in which the strain of mice used was Swiss Webster (SW) mice. The blood collected from several mice by way of the brachial artery, or the guillotine method was pooled. For rabbit blood, the New Zealand White strain of rabbit was used. Blood was collected by puncturing the marginal vein of the ear lobe with a 22 gauge needle. Guinea pig blood was collected from

Duncan Hartley guinea pigs by cardiac puncture. The horse, as well as the cow were bled from the jugular vein into heparin free vacutainers. Fetal Bovine Serum (FBS) was purchased from Sigma Chemicals (Sigma Culture Reagents).

In all cases except with mice and rabbit, the blood was drawn into vacutainers. Pooled mouse blood was collected in centrifuge tubes, and rabbit blood from the marginal vein of the ear lobe was allowed to flow into the centrifuge tubes.

Clotting of Blood:

The blood was incubated at room temperature until

20 clotting occurred, (usually 1 hour to 90 minutes), and centrifuged using a clinical centrifuge (International

Equipment) for 20 minutes at room temperature. The serum that was not used was transferred to vials and stored at -70°C.

Since changes in activity of enzymes were noticed using stored serum, a standard method was chosen for clotting procedures in order to ensure that all samples were being treated under the same conditions. The blood collected into heparin free vacutainers or conical centrifuge tubes was placed on ice until they could be placed in a 30°C water bath. Clotting was allowed to take place at 30°C for one hour. The clotted blood was centrifuged at 2,000 rpm for ten minutes at 4°C. The serum collected was maintained at 4°C until use. The serum that was not used immediately was stored in vials of 1 ml each at -70°C. The glassware, syringes, and all other instruments used were sterilized prior to bleeding.

Enzyme Assays:

Substrates: Synthetic unlabeled leukotrienes LTC4,

LTD4, and LTE4 were purchased from Cayman Chemicals, Ann Arbor, Michigan. The preparations contained 0.1 mg/ml of leukotrienes, dissolved in 0.25 ml of 1:1 ethanol/PBS.

Tritium labeled leukotrienes were purchased from DuPont-NEN products. The specific radioactivity of the leukotrienes was 39.3 Ci/m mol, (2.2 X 106 dpm//1!) in 0.5 ml of 50% 22 ethanol, 0.01 M 2K2P04, at a pH of 6.8. Unlabeled leukotriene (160 p mols) mixed with 22,000 dpm of tritium labeled leukotrienes was used as a substrate.

Hartmann's Solution (Lactated Ringer Solution): Potassium chloride - 0.03 gms Sodium chloride - 0.6 gms Calcium chloride - 0.02 gms Sodium Lactate - 0.31 gms Made up to 100 ml using dd H20.

Assay Mixture:

The assay mixture consisted of 100 ft-l of undiluted serum (unless otherwise mentioned). A stock solution of the leukotriene mix was made up in Hartmann's solution containing 1% Triton X-100 so that each sample received 160 pmols (22,000 dpm) of leukotriene and 25 /*1 of 1% Triton X- 100. This gave an effective dilution of 0.1% Triton X-100 in the assay mixture), the volume of which was made up to

250 ul with Hartmann's solution. Incubation was carried out at 37°C, for specific time periods. The reaction was stopped by the addition of two volumes of methanol at -20°C, containing butylated hydroxy toluene (BHT) as an antioxidant. After the addition of methanol, the samples were mixed well, and kept at -70°C for at least one hour prior to centrifugation, in order to precipitate proteins.

The mixture was then centrifuged at 2,000 rpm for ten minutes at 4°C. The supernatant was transferred to conical centrifuge tubes, evaporated to dryness under vacuum at 23

40°C. Samples were stored at -70°C until HPLC analysis.

The glassware used for the assays were siliconized using Sigmacote (Sigma Chemicals), in order to prevent adherence of LTs to the surfaces, and hence loss of the leukotrienes during extraction.

RBL-1 Cells: Rat Basophilic leukaemia cells were purchased from American Tissue Culture Collection. The cells were maintained in Iscove's modified Dulbecco's medium containing

L-glutamine, in the presence of 10% fetal calf serum.

Stimulation of RBL-1 Cells:

RBL-1 cells were washed twice with 1 ml of serum free media. The cells were resuspended in Phosphate Buffered Saline (PBS, Sigma Diagnostics) at a concentration of approximately 106/ml. The cells were stimulated using lipopolysaccharide (LPS) purchased from Ribi Immunochem

10 ngms/ml, and calcium ionophore (A-23187, Sigma Chemicals) 10 ngms/ml, for 30 minutes at 37°C in the presence of 5 mM calcium chloride (2) . After stimulation, leukotrienes were added, and the samples incubated for 30 minutes at 37°C before lipids were extracted.

Lipid Extraction:

Modified Bligh and Dyer procedure: This procedure was used in preliminary experiments using RBL-1 cells. After the specific incubation time, the cells were pelleted by 24 centrifugation in a clinical centrifuge. To the supernatant, two volumes of methanol were added, mixed well, and the proteins allowed to precipitate at -70°C. The protein precipitate was pelleted by centrifugation at 2,000 rpm at 4°C, and to the supernatant, were added two volumes of chloroform, mixed well for one minute, centrifuged, and the chloroform fraction was separated by centrifugation in a clinical centrifuge. The chloroform fraction was collected in a conical centrifuge tube, evaporated to dryness under vacuum, and stored at -7 0°C until analyzed by reverse phase- high pressure liquid chromatography (RP-HPLC).

Sep-Pak procedure: After protein precipitation using two volumes of methanol, the supernatant was made up to contain

20% methanol, using sodium phosphate buffer (0.1 M, pH 7.4).

The sample was run through a C-18 cartridge (Bakerbond spe, J.T. Baker Inc.), pre-washed with 20 ml methanol and 20 ml water. The cartridge was then washed with 5 ml of 20% methanol in sodium phosphate buffer, followed by 5 ml of water. The leukotrienes in the cartridge were then eluted with 3 ml of 80% methanol in distilled water. This eluent was dried down, and analyzed by RP-HPLC.

Direct evaporation technique: The Sep-Pak procedure resulted in low recovery of leukotrienes in the different fractions. So we developed a method, in which direct evaporation of the supernatant, after protein precipitation, was done. The evaporated sample was stored at -7 0°C, until 25 it was resuspended in solvent and analyzed by RP-HPLC. This procedure involved minimal loss of radioactivity in different fractions, and less loss by adherence to glassware. The presence of other substances with UV absorbance at 280 nm did not interfere with the analysis, since calculation were based only on radioactivity in different fractions.

Reverse Phase High Pressure Liquid Chromatography (RP-HPLC)

Analysis

Solvent: The solvent system used was: acetonitrile: methanol: water: acetic acid (1280 / 800 / 1916 / 4, V/V/V/V). The pH was adjusted to 5.5 using ammonium hydroxide (5). The samples that had been evaporated to dryness were resuspended in 250 pi of solvent and injected into a Nucleosil 5-C18 (25 x 4.6) column (Phenomenex), maintaining a flow rate of 1.0 ml/min. A pressure of 60 psi was applied. The sample was eluted from the column, for a total time of 25 minutes. The column was then rinsed with

100% methanol for 15 minutes. The solvent was then allowed to run through the column for 15 minutes to equilibrate the column before the next sample was injected. The peptidoleukotrienes have a max at 279 nm (38).

The elution times of the different leukotrienes were marked (according to UV absorption peaks at 280 nm) using standard LTC4, LTD4, LTE4, LTF4 and N-acetyl LTE4. In initial 26 experiments, to quantify the relative amounts of leukotrienes present after each assay, the UV absorption peaks of the different leukotrienes at the characteristic elution times were matched with the radioactivity in the corresponding fractions. The fractions were collected using a Gibson model 203 micro fraction collector. The radioactivity in the fractions were then determined in a Beckman 7500 scintillation counter. The different leukotrienes were quantitated based on the percentage of total recovered radioactivity in each peak. In later experiments, a Flo-one Beta In-line radioactive flow detector (Radiomatic) was used in which the UV absorption, as well as the radioactivity of the eluted leukotrienes were simultaneously displayed. RESULTS:

Evaluation of analytical procedures

In the initial experiments with RBL-1 cells, a modified

Bligh and Dyer technique of lipid extraction was compared to a technique using C-18 cartridges (Sep-Pak) (26) and one involving a direct evaporation of the methanol fraction after protein precipitation. The recovery of the leukotrienes by the Bligh and Dyer technique was 58.13% (S.D. 10.43,n=10), while the recovery using C-18 cartridges was 71.48% (S.D. 12.6,n=10), and that by the direct evaporation technique was 89.52% (S.D. 8.61,n=20). The elution profile for a mixture of the different leukotrienes is by RP-HPLC using the solvent system previously described, is shown in Fig. 4.

Leukotriene Metabolism in RBL-1 Cells

3 Initial experiments to study conversion of H-LTC4, standardization of lipid extraction and enzyme assay procedures were done using RBL-1 cells. RBL-1 cells, stimulated by bacterial lipopolysaccharide, in the presence of Ca2+ and A-23187 (calcium ionophore), were seen to

3 3 3 produce both H-LTD4 and H-LTE„ from H-LTC4 (Fig. 5) .

27 28

LTE4

LTC4 LTD4

T T

LTF4 n-acetyl LTE4

Fig. 4. Relative elution times of standard leukotrienes - LTC4, LTD4/ LTE4/ LTF4, and N-acetyl LTE4 .by RP-HPLC 29

Buffer RBL-1

2500

2000

Q 1500 >. o 1000

500

2345 6. 78910 11 12 1314 1,5 16 17,18 1920 t LTC4 LTD4 LTE4 Time (min)

3 Fig. 5. Metabolism of H-LTC4 in the supernatant of RBL- 1 cells. RBL-1 cells were resuspended to a concentration of approximately 10 cells/ml and stimulated with LPS and A 23187 (calcium ionophore) in the presence of calcium. 1ml of the supernatant was assayed for transpeptidase activity and leukotrienes separated by RP-HPLC. Radioactivity was calculated at elution times corresponding to LTC4/ LTD4 and LTE4. Data is expressed as percent radioactivity at different elution times. 30 Leukoreiene Metabolism in Human Serum

After procedures were standardized using RBL-1 cells, human serum from a normal volunteer was used. Table I shows the mean value and standard deviation of values obtained for

the percent conversion of LTC4 after 30 min. incubation and

for LTD4 after 15 min. incubation in six different serum

3 3 samples. Conversion of H-LTC4 or H-LTD4 was not seen in serum at 4°C, Hartmann's solution at 37°C, or following

3 denaturation of proteins(Fig.6,7). H-LTC4 was converted to

3 3 H-LTD4, which was then converted to H-LTE4 as seen by the relative amounts of the different leukotrienes present after 15 and 30 minutes incubation (Fig.8). The experiment was repeated using serum from clotted blood, and plasma from heparinized blood. Heparinized blood showed a decrease in the formation of LTE4, compared to serum (Fig. 9). Thereafter, all experiments were conducted using serum.

The effect of storage of serum on the conversion of

LTC4 and LTD4 was studied using serum stored at 4°C, -20°C and -70°C. Prolonged storage resulted in significant loss of the LTD4 conversion at 4°C, but not at -70°C (Fig. 10). The experiments that followed were all done using fresh serum, or serum that had been stored at -7 0°C immediately after collection of blood.

3 3 The percent conversion of H-LTC4 to H-LTD4 and

3 H-LTE4 was seen to have a linear relationship with the

3 volume of serum used (Fig. 11). The conversion of H-LTC4 31

LTC4 LTD4 Sample no. 1 14 . 82 8 .71 2 12 .50 12 .71

3 16. 64 8 .31

4 14.31 9.74

5 15. 83 10 .56

6 13.51 12.34 Mean 14 . 60 10.39

S. D. + 1.51 + 1 .83

Table I.Mean and S.D. values for percent conversion of 3H- LTC4 conversion (following 30 minutes incubation) and 3H-LTD4 (following 15 minutes incubation) in six serum samples. Fig. 6. Chromatogram showing LTC4 profile after 90 minutes incubation in (A) serum at 4°C (B) Hartmann's solution at 37°C and (C) protein denatured serum. Fig. 7. Chromatogram showing LTD4 profile after 45 minutes incubation in (A) serum at 4° C (B) Hartmann's solution at 37°C and (C) protein denatured serum. 34

Control 15 Minutes 3 0 Minutes

40 LTC4

LTE4

LTD4 o 20

0 2 3 4 5 6 7 8 9 10111213141516171819 20 Time (min)

3 Fig. 8. H-LTC4 metabolism in human serum. 1 ml of fresh 3 human serum was incubated with H-LTC4 for 15 and 30 minutes. The reaction was stopped using 2 volumes of methanol. Leukotrienes were extracted using C-18 cartridges. The recovered leukotrienes were separated by RP-HPLC. Data shows percent radioactivity at different elution times corresponding to the different leukotrienes. 35

Control Clotted Heparinized

.2 25

O —i 2 3 4 5 6. 7 8 9 10 1.1 12 13 14 15 15 17 18 1920 t L LTC4 LTD4 LTE4 Time (min)

3 Fig. 9. Comparison of H-LTC4 metabolized in clotted and heparinized blood, placed at 4 C for approximately 24 hours. Assay procedure was as described in Figure 6. Figure shows relative amounts of LTC4/ LTD4 and LTE4 present in clotted and heparinized blood, as percent of total radioactivity after 60 minutes incubation. 36

Fresh 4° C -20°C

LTE4 LTD4

.2 20

LTC4

o 2345678910 11 12 13 14 15 16 17 18 1920 Time (min)

3 Fig. 10.Comaparison of H-LTC4 metabolism in fresh serum stored at 4°C for 4 days, -20°C for 7 days and -70 C for 7 days after 60 minutes incubation. Procedure was same as described in Figure 8. Data expressed as percent of total radioactivity at different elution times corresponding to the different leukotrienes. 37

100 160

80 c

0 0 1

Volume (ml)

Fig. 11. Relationship between enzyme concentration and 3H- LTC4 metabolism in human serum. Varying amounts of human serum made up to 1ml using phosphate buffered saline, were assayed for transpeptidase activity using procedure described in Fig.8. Data shows percent of LTC4 metabolized (left y-axis) and picomoles of LTC4 converted (right y-axis). 38

100 160

Serum

5 50 80

rio Buffer O 30 60

Time (min)

3 Fig. 12.Relationship between incubation time and H-LTC4 metabolism in human serum. 0.5 ml serum was made up to 1 ml using PBS. Assay procedure as described in Fig 8. Data is expressed as percent LTC4 converted (left y-axis) and picomoles LTC4 converted (right y-axis). 39

1.00 c 1 o CJ

CO o© cE 0.50 - co '5 oC ' O

0.00 0 1000 2000 Substrate Concentration (ng/ml)

Fig. 13.Relationship between substrated concentration and 3 3 H-LTC4 metabolism. Varying amounts of H-LTC4 of known specific radioactivity were used as substrate. Assay procedure was as described in Fig 8. Data expressed as picomoles of LTC4 converted/ml/min. Incubation period was 15 minutes. 40

3 3 (to H-LTD4 and H-LTE4) showed a linear relation to time

(Fig. 12). The conversion of LTC4 also showed a linear increase with respect to substrate concentration (Fig. 13).

Leukotriene metabolism in mouse serum

3 Using H-LTC4 as a substrate, the profile of LTC4 metabolism was compared in mouse and human serum. The results, as shown in Fig. 14, show a great difference. After a 30 min incubation, with 0.5 ml of serum, 50% of the radioactivity was in the LTD4 and LTE4 fractions in human serum, 47% being present as LTD4. Whereas in mouse serum,

3 all the radioactivity in the 15% of converted H-LTC4 was present in the region corresponding to the elution time of

3 LTE4. A time course study using H-LTC4 showed a much higher activity in human serum than in mouse serum (Fig. 15). As this observation suggested a difference in the rate of LTD4

3 metabolism, metabolism of H-LTD4 was compared in human and mouse sera (Fig. 16). The mouse serum was seen to have a

3 much higher activity, 85% of the H-LTD4 being converted in the first 10 minutes of incubation time, while in human serum, only 17% was converted under identical conditions.

3 The metabolism of H-LTD4 in both human and mouse sera was linear with relation to time and substrate concentration

(Figs. 16 and 17). A difference was seen in the rate of conversion with relation to the concentration of enzyme

(volume of serum) used, as shown in Fig. 18. Serial dilution of sera were used in this experiment, because the 41

3 rate of conversion of H-LTD'4 was seen to be much faster than

3 3 that of H-LTC4. The rate of conversion of H-LTD4 did not decrease with increasing dilutions in human serum. This suggested the presence of some factor in the serum that inhibited the enzyme activity in the undiluted form, or in lower dilutions. Heating the sera to 65°C in a water bath for 30 min decreased the activity in both human and mouse serum. The activity in the heated sera of both species was the same (21%) as shown in Fig. 19. The effect of heat inactivated human and mouse serum on unheated human and mouse serum was studied (Fig. 20,21). The results show that while heat inactivated human serum was able to decrease LTD4 conversion in normal human and mouse serum, heat inactivated mouse serum did not show the same effect. 42

Mouse Human

TJ <0 a)1_ >o o

° 25

na « 2a. a

2 3 4 5 5^7 8 9 10 111213 1415 16 17 18 192021 LTC4 LTD4 LTE4 Fraction

3 Fig. 14.Comparison of profiles of H-LTC4 metabolism in human and mouse serum after 30 minutes incubation. Procedure as described in Fig 8. Data expressed as percent radioactivity at different elution times. 43

•a v 53 30 c o G

09 V O oE o Q.

Mouse 0

Time (min.)

3 Fig 15. Effect of incubation time on H-LTC4 conversion in mouse and human serum. 100 ul serum, brought to a total volume of 250 ul with Hartmann's solution and at 0.1% Triton X-100, was incubated in the presence 3 of 160 picomoles H-LTC4 (22,000 dpms) at 37°C. Data expressed as percent of LTC4 converted (left y axis) and picomoles LTC4 converted (right y axis). 44

100 160

.Mouse 120 r (V > c o o (0 80 ® o E o o +Human Q" 40

Time (min)

Fig. 16.Comparison of dipeptidase activity in human and mouse serum. Procedure as described in Fig 15, 3 except H-LTD4 was used as substrate. Data expressed as percent LTD4 converted (left y-axis) and picomoles LTD4 converted (right y- axis). 45

Mouse

e 55

Human

0 0 205 410 [S] (pmoles/0.25ml)

3 Fig. 17. Relationship between H-LTD4 conversion and substrate concentration expressed as picomoles/ml. Experimental procedure as described in Fig. 16, except that different substrate concentrations of varying specific radioactivity were used. Data expressed as picomoles converted/ml/min. Incubation period was 15 minutes. 46

100 160

73 +->O Mouse V > c o O

CO O E o o cC Human

1/1 1/2 1/4 1/8 1/16

Dilution

Fig. 18.Relationship between LTD4 conversion and amount of serum present. 100 ul of 2-fold serial dilutions for human and mouse serum we're used. Experimental procedure as described in Fig. 16. Incubation period was 15 minutes. Data expressed as percent LTD4 converted (left y axis) and picomoles LTD4 converted (right y axis). 47

100 160

80 o

0 Human Human Mouse Mouse Unheated Heated Unheated Heated Serum and Treatment

Fig. 19.Dipeptidase activity in heat treated and untreated human and mouse serum after 30 minutes incubation. The dipeptidase activity was assayed as per procedure described in Fig 16. 48

Normal (+/—) 4- 4- 4- 4- - Heated (ul) 0 25 50 100 100 25 Serum Preparation

Fig. 20.Effect of increasing amounts of heated human serum on the dipeptidase activity in normal human serum after 30 minutes incubation. Data expressed as percent conversion (left y axis), as well as picomoles of LTD4 converted (right y axis) . Different amounts of heat inactivated serum were added to 100 ul of 1/8 dilution of normal human serum brought to 250 ul with Hartmann's solution and 0.1% Triton X-100 and incubated with 160 3 picomoles H-LTD4 (22,000 dpms). 49

100 160 Unheated Mouse

Mouse c •a o > oc 50 80° O Unheated Human • Human eo 0) + Mouse" o -+ E o • Human. o Q"

0 0 0 25 50 100

Heated Serum (ul)

Fig. 21.Comparison of dipeptidase activity in normal human and mouse serum in presence of heat inactivated human or mouse serum after 30 minutes incubation. Procedure as described in Fig 20. Data expressed as percent LTD4 converted (left y-axis) and picomoles LTD„ converted (right y-axis). Leukotriene Metabolism in different animal species

3 3 The metabolism of H-LTC4 and H-LTD4 in human, mouse, guinea pig and rabbit sera were compared by time course studies. No studies were done to compare the conversion of

LTC4 and LTD4 with relation to the volume of serum present, or substrate concentration. Fig. 22 and Fig. 23 show the

3 3 difference in the profile of H-LTC4 and H-LTD4 metabolism in the four species. Fig. 24 shows the relative amount of 3H-

3 LTD4 present in the converted H-LTC4 in the four species after specific incubation times. Fig.25 shows the amount

3 of LTD4 present as a percent of the total H-LTC4 metabolized. The wide variation of this value in the four species and the correlation of this value with the susceptibility to develop anaphylaxis made it necessary to repeat the experiment. In the following experiment, in which fresh serum was used, horse and cow serum and fetal bovine serum (FBS) were included. Fig 26 and Fig. 27 show the time course of LTC4 and LTD4 conversion in the different

3 species. Fig.28 shows the relative amount of H-LTD4

3 present, as a percentage of the total converted H-LTC4, after 15, 60 and 90 min incubation. Fig. 29 shows a comparison of the relative amounts of a radioactive

3 metabolite of H-LTD4 metabolism, which has not been identified. The values mentioned in this experiment are an average of the results from experiments done in duplicate

(Table II). 51

40 64

..Human 30 1 48

-i° Guinea 0 30 60 Pig

Time (min.)

Fig. 22.Comparison of transpeptidase activity in human, mouse, guinea pig and rabbit serum. Procedure as described in Fig 15. Data expressed as percent LTC, converted (left y-axis) and picomoles LTC4 converted (right y-axis). 52

100 160

i^Mouse 80 - 120 •iiRabbit

40 Human o guinea— 40 Rig £L 20

0 10 20 30 Time (min)

Fig. 23.Comparison of dipeptidase activity in human, mouse, guinea pig and rabbit serum. Procedure as described in Fig 16. Data expressed as percent LTD4 converted (left y-axis) and as picomoles LTD4 converted (right y-axis). 53

LTD-4 EZZ2 LTE-4

87.50 140

T>0 70.00 -i—' - 105 CD > c oC o (J '55 52.50 L- <0 0) > c o o £ O o 35.00 o * CL

17.50

0.00 M GP R M GP 20 min. 90 min. Animal

3 Fig. 24.Comparison of H-LTC4 conversion in human, mouse, guinea pig and rabbit serum. The amount of LTC4 converted is expressed as relative amount of LTD4 and LTE4 present after 20 min and 90 min. incubation (percent conversion on left y-axis, picomoles converted on right y- axis) . 54

10C

HUMAN MOUSE G.PIG RABBIT .Animal

3 Fig. 25.Comparison of LTD4 present in the converted H-LTC4 as a percentage of total converted LTC4 after 90 minutes incubation. 55

Horse

100 t. 160r L i Cow oFBS

•a Human o 50 80 to

_o Rabbit _i] Mouse Jfo Guinea 30 60 90 Rg

Time (min)

3 Fig. 26. H-LTC4 conversion in 100 ul of horse, cow, fetal bovine, human, rabbit, mouse, and guinea pig as a time course study. Procedure as described in Fig 15. Data expressed as per cent LTC4 converted (left y axis) and picomoles LTC4 converted (right y axis). 56

FBS 100

fe 50 70 Human »

- 35 ii Guinea Pig

0 15 30 45

Time (min)

3 Fig. 27.Conversion of H-LTD4 in 100 ul of horse, cow, fetal bovine, human, rabbit, mouse, and guinea pig as a time course study. Procedure as described in Fig 16. Data expressed as per cent LTD4 converted (left y axis) and picomoles LTD4 converted (right y axis). 57

V//A 15 min V/////A 60 min KxXH 9 0 min

3 Fig. 28.Relative amounts of H-LTD4 present in 100 ul of horse, cow, human, rabbit, mouse, and guinea pig sera after 15, 60 and 90 minutes. Data expressed as per cent of LTD4 present in the total converted 3 H-LTC4. 58

HH Unknown Y//A LTE—4

100

HUMAN MOUSE G.PIG RABBIT COW HORSE FBS Species

Fig. 29.Relative amount of the unknown radioactive substance present in the total converted LTD„ after 45 minutes incubation. 59

% LTC4 Conversion

Animal

Time Guinea Points Human Mouse Pig Rabbit Horse Cow

15 9.06 0 1.60 0 88.90 78.19 1.22 2.12 0 0 96.92

30 9.05 0 1.63 1.61 98.50 76.74 17.10 0 3.44 3.71 100 71.61

60 34.43 6.68 3.96 7.44 99.05 96.85 34.85 5.28 1.82 13.15 96.60 93.10

90 57.23 5.98 3.35 17.10 98.04 95.75 59.34 3.25 15.70 97.39 100

% LTD4 Conversion

Animal

Time Guinea Points Human Mouse Pig Rabbit Horse Cov

5 2.13 14.63 4.90 54.67 39.79 18.47 1.13 24.23 7.10 55.70 37.92 22.83

15 11.11 63.46 7.10 88.07 67.85 42.47 14.71 63.09 11.59 88.50 75.10 44.38

30 43.45 73.69 8.82 90.00 87.13 68.36 40.00 79.63 14.84 85.00 87.70 67.28

45 52.66 81.36 16.53 97.21 92.70 75.16 46.96 83.08 20.25 95.71 96.40 87.42

"able II. Duplicate values obtained for percent conversion 3 3 of H-LTC4 and H-LTD4 after varying periods of incubation in the sera of the different species tested. DISCUSSION:

Arachidonic acid metabolites have been associated with a variety of inflammatory reactions. Products of the lipoxygenase pathway of metabolism, the peptidoleukotrienes

(formerly referred to as SRS-A) are important mediators in immediate hypersensitivity reactions (24,54). Elevated levels of peptidoleukotrienes were noticed in bronchial asthma and are thought to be responsible for the symptoms in bronchial asthma, which is an immediate hypersensitivity reaction. Elevated leukotriene release from lungs of animals, during experimentally induced anaphylactic reactions have been reported (18,30,53,62). The profile of leukotriene metabolism in different species of animals were studied to see if a correlation exists between the leukotriene metabolism profile, and the relative species susceptibility to develop anaphylaxis.

3 Using H-LTC4 as substrate, supernatant from RBL-1 cells were seen to convert LTC4 to other radioactive substances, which eluted, during RP-HPLC, at times corresponding to synthetic LTD4 and LTE4. The conversion of

3 H-LTC4, was enzymatic since it was not seen to take place in a buffer (Fig. 5).

Lipid extraction and analytical procedures, that gave satisfactory results with RBL-1 cells, were employed to

60 61

3 extract the leukotrienes from human serum. H-LTC4 was metabolized by human serum, with the production of new radioactive products, which eluted with synthetic LTD4 and

3 3 LTE4. Conversion of H-LTC4 or H-LTD4 was not observed in Hartmann's solution at 37°C, serum at 4°C, or in protein

3 denatured serum(Fig. 6,7). The metabolism of H-LTC4 in human serum was dependent on time, amount of enzyme present

(volume of serum) and on the substrate concentration (Fig.

11, 12, 13). In the time course study (Fig. 8), more radioactivity was seen in the LTD4 region with shorter incubation periods. As incubation time increased, the amount of radioactivity decreased in the LTD4 region and increased in the LTE4 region, showing that LTC4 was first converted to LTD4, which was then converted to LTE4. These findings are in accordance with the current knowledge about

LTC4 metabolism; that LTC4 is first converted to LTD4 and that LTD4 is then converted to LTE4 (24, 54). The enzymatic activity in serum was influenced by storage conditions. Prolonged storage at temperatures above

-70°C were seen to decrease the conversion of LTD4 to LTE4

(Fig.10) . A difference in LTD4 conversion was also noticed using heparinized versus clotted blood. When blood was heparinized, the LTD4 conversion was the same as in clotted blood, when assayed immediately; however, when heparinized blood was stored overnight at 4°C, prior to separation of plasma, the amount of LTD4 converted was seen to be reduced 62

2+ 2+ 2+ (Fig. 9). The LTD4 conversion requires Ca , Mg , and Mn for optimal activity (39,52). The presence of substances like L-cysteine and dithiothreotol, which remove divalent cations from the medium are known to inhibit the LTD4 conversion (39). Subsequent addition of these cations at 1 mM concentration was seen to restore the lost activity (39). Although the anticoagulant effect of heparin is not due to the removal of Ca2+, it is possible that it is acting as an inhibitor of LTD4 conversion because it has the property of activating protease inhibitors, which in turn prevent the conversion of prothrombin to thrombin (70).

Initially, leukotriene metabolism in serum of normal

Balb-C mouse was compared with that in human serum. The profile in normal mouse serum was seen to be very different from that in humans (Fig.14,15). After 30 minutes incubation, 0.5 ml of normal human serum showed 67% conversions, with 34.6% recovered as LTD4 and 15.1% as

LTE4. The mouse serum showed only 13.4% conversion, and

3 all the radioactivity of the metabolized H-LTC4 was recovered as LTE4. It was presumed from these results, that the formation of LTD4 must be a much slower process in mouse than in humans, and that the LTD4 formed was immediately

3 converted to LTE4. Subsequent experiments with H-LTD4 as substrate, showed that the amount of LTE4 formed during LTD4 conversion was 4.2 times more in mouse than in humans

(Fig.16). These findings correlate with results of 63 experiments by Applegreen and Hammarstrom (4), who found

3 that 20 minutes after intravenous injection of H-LTC3 into mice, the radioactivity was recovered as LTC3 in liver and

3 other organs. H-LTE3 was detected only in the lung, after

3 60 minutes. Recovery of H-LTD3 was not reported in any tissue. LTC3 has been reported as having similar biological activity and enzyme specificity as LTC4 (21); therefore the profile of LTC3 metabolism should reflect that of LTD4.

Since LTD4 is biologically the most potent peptidoleukotriene, the absence of LTD4 accumulation in mouse serum, correlates with the reported resistance to the development of anaphylaxis in the mouse (35,45,69).

The conversion of LTD4 was studied in mouse and human

3 sera, in the same way as the LTC4 conversion, using H-LTD4 as substrate. The rate of conversion of LTD4 increased with time, as well as with substrate concentration (Fig.16 and

17). However, the rate of reaction did not decrease in a linear manner, with increasing dilutions (Fig. 18). The rate of conversion increased up to a dilution of 1/8, and then declined. This observation implied that the serum contained another factor, the presence of which was reducing

LTD4 conversion, in the undiluted form. Initial dilutions could have diluted the effect of this factor, leading to an increase in the LTD4 conversion.

The presence of an inhibitor of LTD4 conversion has been mentioned briefly, by Raulf and coworkers in 1985 (51). They suggested that a rapid decline in LTD4 conversion in supernatant of stimulated RBL-1 cells was probably due to the presence of inhibitors. An attempt was made to study the nature of this "inhibitor" in serum. The enzyme activity in the serum was destroyed by immersing serum in a 65°C water bath for 30 minutes (Fig. 19). This temperature was chosen, because temperatures above 65°C solidify the serum, due to coagulation of serum proteins, making it unfit for enzyme assay. Addition of heat inactivated human serum to unheated human serum was seen to decrease the LTD4 conversion by the unheated serum, in a concentration dependent manner (Fig.20). The effect of heat inactivated human and mouse serum, on unheated human and mouse serum, was compared. The heat inactivated human serum, seemed to contain larger amounts of the "inhibitory substance'', as shown by the dramatic reduction of activity in normal mouse serum (which showed high rate of LTD4 conversion), as compared to the reduction of activity in human serum. Heat inactivated mouse serum did not have as much an inhibitory effect on mouse serum, or on human serum

(Fig.21). Based on the results of this experiment, human serum was thought to contain a higher concentration of an unknown heat stable substance, which decreased the rate of conversion of LTD4. This "inhibitory factor", could have

3 increased the accumulation of H-LTD4 in human serum.

Absence, or a less amount of this substance in mouse serum might contribute to the higher resistance offered by mice to the development of anaphylaxis. Further studies, leading to characterization of this "inhibitory factor" are now in progress.

The interesting results obtained by comparing human and mouse serum, with respect to leukotriene metabolism, and the correlation of these results with the anaphylactic reaction in these two species, lead us to conduct similar experiments on sera of other animal species. Guinea pigs and rabbits are reported to develop severe anaphylactic reactions (9). Anaphylactic reactions in horse and cattle have also been described (8). In an effort to identify a basis for the

3 3 differences in hypersensitivity reactions, H-LTC4 and H-

LTD4 were used as substrates to study the profile of leukotriene metabolism in human, mouse, guinea pig and rabbit serum. The profile of LTC4 conversion different in all four species (Fig. 22). It was highest in human serum, 32% being converted by 100 ul of serum after 60 min incubation. Rabbit and mouse sera showed a similar profile,

6% and 4% being converted after 60 min, while guinea pig serum showed least activity, 0% LTC4 being converted after

60 min. LTD4 is the most potent of the peptidoleukotrienes, with respect to biological activity. Therefore the relative amount of LTD4 present as a percent of the total converted

3 H-LTC4 was determined (Fig. 24 and 25). Larger amounts were present in human, guinea pig and rabbit sera, 25, 65 66 and 72% being present respectively. In the mouse serum,

3 3 only 4% of the converted H-LTC4 was present as H-LTD4. The difference in the relative amount of LTD4 present suggested

3 a difference in the metabolism of LTD4. Using H-LTD4 as a substrate, LTD4 metabolism in the four species was compared (Fig.23). The mouse serum showed highest activity, which accounted for the negligible amounts of LTD4 accumulation in mouse serum. The susceptibility to the development of anaphylaxis, in the different species described by earlier workers (9,50), seemed to be related to the rates of synthesis and metabolism of LTD4; i.e., the lower the rate of LTD4 synthesis, or the higher the rate of LTD4 conversion, less prone was the animal to develop a severe anaphylactic reaction.

The above experiment was done using sera stored at -70°C immediately after collection. Another experiment was designed using fresh serum from each species. In this experiment, which was done in duplicate, the serum was placed at 4°C (for 1 hour maximum time) until the assay was done. Sera from horse, cow and fetal bovine were also included. The mouse serum in this experiment came from

Swiss Webster (SW) mice, whereas in the previous experiment it was collected from Balb-C mice. Sera from horse, cow and

FBS showed maximal activity, more than 50% of LTC4 being converted in the first 15 min of incubation (Fig.26).

Metabolism in human, rabbit, guinea pig and mouse sera showed similar patterns as those described above. However, there were differences in the relative amounts of LTD4 produced. The relative amounts of LTD4 present after 15, 60 and 90 min. incubation are shown in Fig. 28. In mouse serum, 100% of the converted LTC4 after a 60 min incubation was seen as LTD4, in contrast to the complete conversion to

LTE4 in the previous experiment. Nevertheless, only 6.7% of

3 the H-LTC4 was metabolized. Moreover, a high rate of LTD4 conversion was observed in the mouse serum, which could could lower the LTD4 level as soon as it arises. A significant decrease in the LTD4 levels was seen after 90 min. This difference seen between the two experiments, may be due to a strain difference. Since development of anaphylaxis is thought to be genetically controlled (58), it could be that a strain to strain difference exists in the susceptibility to develop anaphylaxis. A strain difference in asthma models in rats has been reported (37). Experiments dealing with induction of anaphylaxis in different strains of mice are necessary to confirm this view.

3 The addition of H-LTC4 to rabbit serum resulted in

3 the accumulation of a large amount of H-LTD4 even after 90 minutes incubation (Fig. 28). This correlated with the susceptibility of the animal to develop anaphylaxis. In the presence of a high rate of LTC4 conversion, a slower rate of

LTD4 conversion would be expected, in order to maintain high

3 levels of LTD4 even after 90 minutes incubation with H-LTC4. 68

3 Surprisingly, rabbit serum showed a high rate of H-LTD4 conversion (Fig.27). This observation was different from that in the previous experiment. In human serum, a large amount of LTD4 was present after 60 and 90 min incubation

(Fig.28). This appeared to result from a high rate of LTC4 metabolism and a lower rate of conversion of LTD4 to LTE4.

This metabolic profile might maintain a high level of LTD4 for prolonged periods of time. This pattern of leukotriene metabolism might, therefore contribute to the prolonged symptoms of anaphylaxis in humans, ( eg., the symptoms of bronchial asthma). The slow rate of metabolism of LTD4 might account for the high amounts of LTD4 in the supernatant from alveolar macrophages in asthmatics individuals, as compared to those from normal individuals, reported by Damon and coworkers (17).. However,

Schwartzberg and coworkers (56), and Isono and coworkers

(26) found only LTC4 in the serum during an acute asthmatic attack in children. These findings suggest that during an acute asthmatic attack, the metabolism of leukotrienes in the lung might be different from that in the blood.

The LTC4 conversion as well as the LTD4 conversion activity in guinea pig serum seemed to be the lowest of all sera tested (Fig. 26,27). Although the rate of conversion of LTC4 was low, the slow rate of LTD4 conversion might be

3 responsible for accumulation of the converted H-LTC4 as

LTD4. These observations correlate with those of in vivo 69 experiments conducted by Keppler et al (30), where LTD4 was the main metabolite in bile following anaphylactic shock in the guinea pig. Clancy and coworkers (15) have reported

LTD4 as the main metabolite in the supernatant of guinea pig lung tissue after incubation with LTC4. De Nucci and coworkers (18) have identified LTD4 as the main metabolite in perfused lung tissue, after antigen challenge. Guinea pig and mouse sera showed a similarity in the profile of 3H-

LTC4 metabolism. On comparing the relative amounts of LTD4

3 3 following H-LTC4 metabolism, 100% of the metabolized H-LTC4 was recovered as LTD4 after 60 minutes incubation. However, the rapid rate of LTD4 conversion in mouse serum brought about a reduction in the 3H-LTD4 levels after 90 minutes incubation (Fig 28). This suggests that LTD4 conversion is more important than LTC4 conversion in controlling -the accumulation of LTD4.

Although guinea pig and rabbit sera are similar in the relative amounts of LTD4 present, the anaphylactic reaction is different in the two species. In guinea pig, the anaphylactic reactions mimic that in humans, and the respiratory distress is similar to that seen in bronchial asthma in humans (50). These symptoms are due to narrowing of bronchioles due to smooth muscle constriction. Although respiratory distress occurs in rabbit, it is not due to bronchoconstriction, but rather due to constriction of the pulmonary artery. This has been shown by experiments in which a rise in pulmonary arterial pressure was demonstrated (16). The pulmonary artery of the rabbit has a more developed smooth muscle layer, whereas the bronchioles have less smooth muscle than in the guinea pig. After fatal anaphylaxis, a difference in lung pathology supports the fact that in rabbit, death is due to cardiac failure due to raised pulmonary arterial pressure, whereas in guinea pig it is due to bronchoconstriction (50). For these reasons, the guinea pig is known to be a better experimental animal model for bronchial asthma.

In horse and cow serum, both LTC4 and LTD4 conversion were seen to be high, reaching almost maximal levels after 15 min incubation (Fig.26,27). Although large amounts of

3 H-LTD4 are formed in the initial stages, these levels decline rapidly due to increased LTD4 conversion. This observation correlates with the description of the anaphylactic reaction in horse and cattle (8), where the symptoms of respiratory distress develop within 8-15 minutes, and then subside spontaneously. The horse and cow serum appeared to be similar to human serum in that significant amounts of LTD4 were present after 15 min incubation. This might be one of the factors responsible for the similarity in symptoms of anaphylaxis between humans, horse and cattle (50).

The relative amounts of LTD4 present were expressed

3 as a percentage of the total converted H-LTC4. The radioactive substances other than LTD4 were not always confined to the LTE4 region. In certain species, as in horse, and in FBS, an additional radioactive peak was seen immediately after the elution of LTE4. The elution time of this metabolite did not coincide with that of standard preparations of LTC4, LTD4, LTE4, N-acetyl LTE4, or LTF4. The formation of this unknown substance did not appear to be related to the rate of LTD4 conversion reaction (Fig 29) , as it was not seen in rabbit and mouse serum which showed high rate of conversion of LTD4. Hammarstrom (23) has reported the presence of eight LTC4 metabolites in the feces of rats

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