CLINICAL REVIEW 167 Procalcitonin and the Calcitonin Gene Family of Peptides in Inflammation, Infection, and Sepsis: a Journey from Calcitonin Back to Its Precursors

CLINICAL REVIEW 167 Procalcitonin and the Calcitonin Gene Family of Peptides in Inflammation, Infection, and Sepsis: A Journey from Calcitonin Back to Its Precursors

K. L. BECKER, E. S. NYLE´ N, J. C. WHITE, B. MU¨ LLER, AND R. H. SNIDER, JR. Veterans Affairs Medical Center and George Washington University (K.L.B., E.S.N., J.C.W., R.H.S.), Washington, D.C. 20422; and University Hospitals (B.M.), CH-4031 Basel, Switzerland

Calcitonin (CT) is a hormone that received its name be- Immature and mature CT cause of its secretion in response to induced hypercalcemia It had been found that immunoreactive CT was present in and its hypocalcemic effect (1). It was shown to originate multiple heterogeneous forms in MTC tissue as well as in the from the thyroid gland (2). More specifically, the hormone serum of patients with this tumor (20–24). Consequently, it was revealed to be located within the thyroidal parafollicular became apparent that when this peptide was measured with cells, interspersed within and about the follicular epithelium antisera recognizant to different epitopes the values varied (3–5). Subsequently termed C cells, they occur primarily in according to the antiserum and the immunochemical heter- the central region of each lobe of the human thyroid gland ogeneity (25). The phenomenon of heterogeneity was then (6, 7). These cells, which have CT-containing secretion gran- further clarified by a series of studies which demonstrated ules, are neuroendocrine. Embryologically, they originate that CT is biosynthesized as part of a larger prohormone, from the neural crest and migrate to the ultimobranchial procalcitonin (ProCT) (Fig. 1) (21, 23, 25–27). glands (8). In mammals, the ultimobranchial glands fuse The term “mature” hormone has been used to indicate a with the thyroid gland. bioactive hormonal peptide that has been derived from a It was the demonstration that medullary thyroid cancer larger precursor prohormone. This prohormone may be less (MTC) was a malignancy of the C cells (5, 9) that eventually active, inactive, or characterized by an activity that differs led to the isolation of human CT from this tumor and the entirely from the mature hormone. Not uncommonly, much determination of its structure (10, 11). Simultaneously, the of the bioactivity of a mature hormone may be linked to an amino acid sequence of porcine CT was determined (12). amidation that occurs at its carboxyl end. Within ProCT, CT Later, the development of immunoassays of serum CT in is in a nonamidated, immature 33-amino acid form, termi- humans led to the observation that the level of this hor- nating with a glycine (28). It then undergoes posttransla- mone was increased in the serum of patients with MTC tional processing that results in production of several addi- (13–15) and to the demonstration that these levels were tional free peptides as well as mature CT (29–31). further augmented after iv calcium and/or pentagastrin All species of mature CT contain 32 amino acids, with a administration (13, 16, 17). These findings had a great disulfide bridge at the amino terminal end (between amino impact on the clinical diagnosis, the evaluation of efficacy acid positions 1 and 7) and a proline at the carboxyterminal of surgical extirpation, and the follow-up monitoring of end; hence, for the purpose of clarity in this manuscript, the MTC. Although RET germline mutation testing has re- term CT(1–32) will be used specifically to refer to this pep- placed CT for the purpose of determining the presence of tide. Among the various species of CT(1–32), the amino acid carriers of this tumor associated with multiple endocrine sequence of the peptide tends to be well conserved within the neoplasia type 2 (18, 19), the measurement of serum CT has amino acid ring structure at the amino terminus, but there are become and has remained the classical clinical marker for differences elsewhere within the molecule (32–34). At the MTC. carboxyl terminus of the CT(1–32), the proline is amidated (35, 36). Importantly, both the ring structure and this ami- Abbreviations: CCP-I, 21-Amino acid CT carboxyterminus peptide I; dated proline are essential for the full expression of the CGRP, CT gene-related peptide; CT, calcitonin; CTpr, CT precursors; known bioactions of this hormone. LPS, lipopolysaccharide; MTC, medullary thyroid cancer; NO, nitric The accurate quantification of the free CT(1–32) peptide oxide; NProCT, 57-amino acid sequence at the amino terminus of ProCT; requires the selective detection of the amidated carboxyl PNE, pulmonary neuroendocrine (cell); ProCT, procalcitonin; SCLC, small cell cancer of the lung. terminal portion of the molecule, thus excluding the nonamidated 33-amino acid immature CT, which is found within JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the en- some of the larger molecular weight precursors. Such com- docrine community. mercially available assays were not developed until the late

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FIG. 1. Schematic representation of ProCT and the other CT precursors (CTpr) derived from this prohormone (i.e. NProCT, CT-CCP-I, and CCP-I). The mean concentrations of these peptides in normal serum is indicated. Note that there is appreciably more free NProCT in the serum than CT(1–32). In sepsis, the principal elevations involve the intact ProCT, free NProCT, and free conjoined CT-CCP-I peptide. Sequencing reveals that in sepsis, the ProCT may lack the first two amino acids of the aminoterminus of the molecule, presumably due to enzymatic hydrolytic aminoterminal truncation (110), and perhaps other cleavage forms are present as well (111). The comparative extent to which any one of these peptides is increased varies among patients. Levels of the free CCP-I peptide also increase but to a lesser extent. In sepsis, serum CT(1–32) concentrations are undetectable, normal, or only slightly to moderately elevated (data from Ref. 30).

1980s; they use a double-antibody method: one antibody ever, these mice exhibited an increased calcemic response reacts selectively with the amidated region and the other and a greater bone resorption in response to exogenous PTH, with a different portion of the molecule (most commonly the perhaps due to the absence of an otherwise inhibiting effect midportion). Thus, these assays do not cross-react with im- of CT(1–32) on bone resorption. Surprisingly, these knockout mature CT (37–39). In this regard, it is important to empha- mice manifested a markedly increased bone formation; also, size that most CT studies in the literature relating to phys- in contrast to wild-type mice that lose bone mass after ovari- iopharmacologic manipulations as well as such influences as ectomy, they maintained their bone mass. These findings age, gender, pregnancy, and hormonal milieu were not doc- suggest that the CT/CGRP-I gene product may somehow umented with these specific assays. regulate bone formation, either directly or indirectly. Further studies of these interesting observations are needed to de- Physiologic actions of CT termine whether this action is related to CT(1–32), CGRP-I, Hundreds of studies of the possible role of CT(1–32) have or both acting conjointly, and also whether it is species spe- been performed. The great bulk of in vitro and in vivo inves- cific. Additional studies should also determine whether the tigations have involved laboratory animals, some with prior induced knockout results in a compensatory overexpression parathyroidectomy and some without. Often the species of of the gene that gives rise to CGRP-II, which, as a result, may CT(1–32) used in these experiments were other than human conceivably modulate or modify the resultant phenotype. (e.g. salmon, porcine, eel), the amino acid sequences of which The classic and best-studied action of CT(1–32), which differ. Furthermore, pharmacologic, not physiologic, doses appears to occur generally throughout the mammalian spe- often were employed. As a result, many actions have been cies, is the action on the osteoclast (34, 47, 48). Acutely, this incorrectly imputed to this peptide. Known or alleged bio- hormone alters the osteoclast sensitivity to ambient calcium logic actions of CT(1–32) have been reviewed elsewhere (31, and induces quiescence of osteoclast motility and a retraction 33, 40, 41). of the pseudopods that is associated with a cessation of Although seemingly relevant effects have been observed membrane ruffling. The peptide also inhibits the elaboration in blood, bone, kidneys, and the respiratory, gastrointestinal, by the osteoclast of acid phosphatase, carbonic anhydrase II, embryogenic, and central nervous systems (40, 42–45), the focal adhesion kinase, and osteopontin. Possible anabolic function of CT(1–32) in humans remains enigmatic (41). The effects of CT(1–32) on the osteoblast have been reported (49) hormone is not confined to the thyroid gland, and it is im- but require further documentation. The overall impact of the possible to extirpate all cells producing this peptide (see osteoclastic inhibition is to decrease bone resorption (50). below). However, the recent development of a knockout Nevertheless, neither the diminution of serum CT(1–32) oc- mouse in which the coding sequences for both CT(1–32) and curring subsequent to thyroidectomy nor the marked excess CT gene-related peptide (CGRP)-I were deleted have pro- of serum levels of CT(1–32) that occurs in patients with MTC, vided important information (46). In these animals, no birth appear to be associated with alterations in serum calcium or defects or difficulty with procreation were noted, and serum noticeable decreases or increases of bone mass (51). Perhaps levels of basal calcium-related values were normal. How- the major function of CT(1–32) is to combat acute hypercal-

Downloaded from jcem.endojournals.org by on October 15, 2007 1514 J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 Becker et al. • Clincal Review cemia in emergency situations and/or to conserve calcium ProCT and CT precursors stores during growth, pregnancy, and lactation. CT(1–32) is biosynthesized from the polypeptide precur- The osteoclast has a CT receptor, as do other cells else- sor, ProCT. This 116-amino acid prohormone is comprised of where, e.g. monocytes, kidney, brain, pituitary, placenta, three constituent peptides: a 57-amino acid sequence at the prostate, testis, lung, and liver (52–55). The CT(1–32) receptor amino terminus (NProCT); the centrally positioned imma- has been cloned (56–58). In the human, there is a polymor- ture CT that contains a terminal glycine; and a 21-amino acid phism of this receptor (59), which may clinically influence CT carboxyterminus peptide I (CCP-I) (Fig. 1) (28). Subse- bone density and quality (60), and these different isoforms quent enzymatic posttranslational processing yields several may also have other functional implications. Stimulation of peptides (31, 73, 74); in addition to CT(1–32), the serum of the CT(1–32) receptor induces increased cAMP and an in- normal persons contains intact ProCT, free NProCT, free creased cytosolic free calcium concentration, accompanied CCP-I, and the free conjoined CT-CCP-I peptide. Interest- by activation of the MAPK pathway (61). ingly, the normal molar concentration of circulating NProCT is 2-fold higher than that of CT(1–32) (4.15 fmol/ml vs. 1.84 fmol/ml) (30). Because these peptides are relevant to and Therapeutic effects of CT(1–32) precede the biosynthesis of CT(1–32), they have been given Although the usage of CT(1–32) as a drug has greatly the collective appellation CT precursors (CTpr) (75, 76). The diminished, it continues to have a therapeutic role, princi- extent to which CTpr may have physiologic functions is pally in osteoporosis and Paget disease. The commercially under study (see below). used drug is the amidated, synthetic salmon preparation; in the United States, human CT(1–32) has become an orphan CTpr in MTC drug. For osteoporosis, it is commonly agreed that bisphos- phonate therapy is more effective than CT(1–32) and is An appreciable number of conditions are associated with currently the preferable therapy (62). However, salmon increased serum levels of CTpr (Table 1). Although it has been known for many years that normal thyroid gland, MTC CT(1–32) has been well demonstrated to increase bone den- tissue, and the serum of MTC patients contain large amounts sity and decrease fracture rate, especially in the vertebrae of ProCT as well as its component peptides (22, 77), the (63–65); adverse effects are minimal. For osteoporosis, the potential clinical utility of CTpr as a serum marker for MTC nasal or sc route can be used. Bisphosphonates also have has very rarely received attention. In this respect, initially it replaced CT(1–32) for the therapy of Paget disease (66, 67); was found that the carboxyterminal region of ProCT (cor- nevertheless, sc salmon CT(1–32) still may be useful in the responding to CCP-I, also termed katacalcin) was secreted occasional patient who cannot tolerate high doses of bisphos- into the medium of MTC cultures as well as into the serum phonates (68). In the United States, the nasal CT(1–32) prep- of patients with this tumor in a calcium-dependent manner aration is not approved for Paget disease. As an acute or (78). Subsequently an assay for CCP-I was evaluated as a subacute therapy for hypercalcemia, the sc or im adminis- serum marker for MTC (79). It was later reported that the tration of salmon CT(1–32) is not a reliable procedure; the NProCT cleavage product of ProCT also was a potential treatment of choice is adequate hydration and iv bisphos- marker for this tumor (80). Recently, in a study of MTC phonates (69). Various possible antinociceptive effects of patients (81), a sensitive and rapid (3-h) two-antibody sand- CT(1–32) have been described (70). For example, in patients wich assay that quantifies both intact ProCT and CT-CCP-I with painful osteolytic metastases, symptomatic therapy (82) was compared with an assay that is specific for CT(1–32). may be beneficial (71); however, the limited evidence in the CTpr were found to be universally present in the serum of literature does not support its use for this purpose (72). patients with MTC. They were increased whenever CT(1–32)

TABLE 1. Clinical conditions in which serum CTprs are increaseda

Sepsis with or without documentation of bacterial infection; sepsis-related conditions, such as pancreatitis, severe burns, polytrauma, heatstroke, marked systemic bacterial infection as may occur in pneumonia or pyelonephritis; occasional patients with marked systemic viremia or fungal infection; and severe malaria Medullary thyroid cancer Aspirational or inhalational pneumonitis Adult respiratory disease syndrome (ARDS) Pulmonary neuroendocrine hyperplasia as occurs in chronic obstructive pulmonary disease or smoking-related chronic bronchitis Small cell cancer of the lung Non-small cell lung cancer (probably due to admixture of malignant small cells, and/or to smoking-related pulmonary neuroendocrine cell hyperplasia) Carcinoid tumor Other neuroendocrine tumors (pheochromocytoma, pancreatic islet cell tumor) Other, seemingly non-neuroendocrine malignancies, such as breast cancer a In these listed conditions, using a sensitive assay (76), CTpr may be increased to enormous levels in the serum, e.g. ranging from 1,000 to 100,000 pg/ml in patients with sepsis, or, only minimally increased, e.g. 200 pg/ml in pulmonary neuroendocrine hyperplasia [normal, less than 76 pg/ml (12 fmol/ml)]. In sepsis and septic-related conditions, CT(1–32) usually is normal or only minimally increased. In contrast, the high serum CTpr that occurs in medullary thyroid cancer, small cell carcinoma of the lung, carcinoid, and some other neuroendocrine tumors is nearly always accompanied by a marked increase of serum CT(1–32).

Downloaded from jcem.endojournals.org by on October 15, 2007 Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1515 was increased and, on a pg/ml basis, exceeded CT(1–32) of macrophages, altered capillary function, transudation of levels by about 10-fold. Also, both markers responded to serum into the tissues, and the release of various humoral pentagastrin. Whether CTpr are more sensitive markers for substances (Table 2). the presence of MTC or might be more useful prognostically There are a variety of distinct conditions that manifest remains to be determined. systemic inflammation (e.g. severe burns, pneumonitis or other marked local infections, bacteremia, endotoxemia, CTpr in several other clinical disorders trauma, heat stroke, and pancreatitis). Typically any of these Other neuroendocrine tumors, such as small cell cancer of conditions may lead to a clinical syndrome that has been the lung (SCLC), carcinoid, pheochromocytoma, and pan- termed the systemic inflammatory response syndrome, creatic islet tumors may exhibit increased serum CTpr. In which is defined by varying combinations of fever or hypo- contrast to MTC, in these conditions, the serum CTpr/ thermia, tachypnea, tachycardia, and polymorphonucleocys- CT(1–32) ratio is further increased, probably because these tosis or leukopenia (94, 95). Associated with these manifes- lesions lack sufficient posttranslational enzymatic capability tations, there is a variable local as well as systemic increase (30, 83, 84). In the case of SCLC and carcinoid tumor, the of many cytokines and other hormonal messenger molecules e.g. ␣, IL-1␤, IL-6, interferon-␥, arachidonic acid deriv- occurrence of increased serum CTpr is very frequent. Using [ TNF atives, cortisol] (Table 2). Some of these substances, acting in an immunoassay that is reactive to the midportion of CT [and a hemocrine and/or paracrine manner, are protective to the hence detects the presence of the ProCT and free CT-CCP-I host; some may be harmful, and some may be either bene- molecules as well as CT(1–32)], there was found to be an ficial or damaging, depending on their concentrations, their association between the levels obtained and the clinical timing, or the ambient humoral milieu. Alternatively, the course of the tumor (85). Similarly, levels usually decreased increases of some of these substances may be epiphenomenal concomitantly with radiotherapy and/or chemotherapy. and exert no relevant bioeffects. Also, decreased levels corresponded to clinical remissions. The clinical term, sepsis, has been used to indicate a sys- Interestingly, SCLC is thought to originate from the same temic inflammatory response syndrome in which bacteria or precursor cell as does the normal pulmonary neuroendocrine microbial products have been shown or suspected to be the (PNE) cell, a cell that contains large amounts immunoreac- etiology. In some cases, infection cannot be documented tive CT (42, 86, 87). However, lung cancers other than SCLC because microbial culture does not reveal a pathogenic mi- may contain and secrete immunoreactive CT. In such pa- crobe. It is likely that in some of these culture-negative cases, tients, these peptides may originate from the tumor, admixed the sepsis is indeed due to microbes, but the methods of SCLC cells, or adjacent noncancerous PNE cells that are detecting them are not completely reliable and, therefore, the known to become hyperplastic in response to chronic ciga- pathogen may not be identified. Moreover, in other cases, rette smoking (88, 89). Heterogeneity studies that have been toxic byproducts of the pathogen may be responsible for the performed in such cancer patients also reveal a large CTpr/ syndrome. For example, the translocation through the gut CT(1–32) ratio (83). wall of toxins [e.g. endotoxin (lipopolysaccharide, LPS)] from Some noncancerous conditions that are associated with bacteria normally inhabiting the gastrointestinal tract may be increased levels of serum CTpr appear to be explicable, all or the cause of the sepsis (96, 97). in part, on the basis of hypersecretion or hyperplasia of the In sepsis, the patient is not ill principally because of the PNE cells [i.e. chronic bronchitis (e.g. smoking, occupational, initial injury or infection but because of a humoral and/or cystic fibrosis), chronic obstructive pulmonary disease, acute cellular overreaction of the host. The unrestrained or unbal- inhalational burn injury, acute chemical pneumonitis, and anced cytokine and humoral response in this illness may tuberculosis] (84, 90). Furthermore, the kidney plays an im- progress sufficiently to cause multiple organ failure, char- portant role in the metabolism of CT(1–32) (91), and renal acterized by varying degrees of severe occurrences, such as disease often is associated with increased serum immuno- myocardial insufficiency, hypoperfusion, shock, coagulopa- reactive CT levels, much of it consisting of CT-precursor thy, respiratory failure, hypoxemia, renal failure, and coma. peptides (24, 92, 93). Sepsis is the 11th leading cause of death in the United States. Lastly, as emphasized in the present review, extraordinary Approximately half of the fatalities in intensive care units increases of serum CTpr occur in patients with severe in- result from this condition. The incidence of sepsis is increas- flammation, systemic infection, and sepsis. Indeed, CTpr ing (currently approximately 750,000 cases per year), and the serum levels can be used as markers for the presence and mortality remains unchanged (approximately 30%, with severity of these conditions. Furthermore, in these condi- rates up to 75% in septic shock) (98). tions, high levels of CTpr appear to play a harmful role, and their immunoneutralization offers the potential of effective therapy. CTpr as markers of inflammation, systemic infection, and sepsis. An initial publication in 1983 first called attention to in- CTpr in inflammation, systemic infection, and sepsis creased serum levels of immunoreactive CT in patients with the staphylococcal toxic shock syndrome, a severe form of Pathophysiology. Inflammation, a highly complex phenome- sepsis (99). The assay used an antiserum that was not selec- non that may be beneficial and/or detrimental to the host, is tive for the amidated carboxyl terminal portion of CT(1–32) a reaction to a large variety of injuries. Inflammation can be and hence would also bind to the immature CT within the local or systemic. It is characterized by vasodilation, attrac- ProCT and CT-CCP-I molecules. Gel filtration studies dem- tion of polymorphonuclear cells and lymphocytes, activation onstrated that this immunoreactive CT was of large molec-

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TABLE 2. Schematic illustration of events and humoral factors following exposure to a variety of insults that trigger an inflammatory responsea

a Several of these factors have multiple and/or variable roles, and some may be either anti- or proinflammatory. In addition, many other humoral factors have been identified. For further documentation and elaboration, see Refs. 183–186. ADM, adrenomedullin; AP-1, activator protein-1; AVP, arginine vasopressin; CRP, C-reactive protein; HMG-1, high mobility group-1; IFN, interferon; IL-ra, IL receptor antagonist; LTs, leukotrienes; MIF, monocyte migration inhibitory factor; MOF, multiple organ failure; NF-␬B, nuclear factor-␬ B; PAF, platelet activating factor; PAMPS, pathogen-associated molecular patterns; PGs, prostaglandins; PTX, pertussis toxin; RANTES, regulation on activation normal T-expressed and secreted; ROI, reactive oxygen intermediates; TBs, thromboxanes; TNFr, TNF receptor. ular weight, now known to correspond to ProCT and CT- and the routine laboratory tests (e.g. an abnormal leukocyte CCP-I. This paper provided the inspiration for multiple count, elevated serum C-reactive protein, or positive bacte- subsequent studies of CTpr in inflammation, systemic infec- riologic studies) may be nonspecific or may not occur at all. tion, and sepsis. Furthermore, the classical associated proinflammatory cyto- The traditional clinical signs of severe infection (e.g. fever kines of severe inflammation, systemic infections, and sepsis or hypothermia, tachycardia, tachypnea, or hypotension) (i.e. TNF␣, IL-1␤, or IL-6) commonly are increased in the

Downloaded from jcem.endojournals.org by on October 15, 2007 Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1517 serum only transiently or intermittently. In contrast, serum present in the sera of sepsis patients, there appears to be levels of CTpr are very frequently increased, sometimes at- universal agreement that both ProCT and multiple fragments tain levels several thousand-fold normal, and these high of ProCT are present. Interestingly, in all these sepsis and levels often persist for long periods of time. Moreover, the sepsis-like conditions, CT(1–32) remains undetectable or nor- levels often correlate positively with the severity of the con- mal or slightly to moderately elevated (30, 100, 102) (see dition and mortality. Indeed, after the first systematic study below). of sepsis due to severe bacterial infection (100), many clinical There are four assays that have been created to measure studies have documented the considerable utility of serum serum CTpr. Currently none of these assays are commer- CTpr to identify and follow the course of this illness and cially available in the United States. The authors developed sepsis-like conditions (Fig. 2) (100–102). a single-antibody RIA for NProCT, which quantitates ProCT Thus, serum CTpr have been shown to be extremely useful and the free NProCT peptide (as well as ProCGRP and markers in sepsis, whether blood cultures are positive or NProCGRP) (76, 101). This sensitive assay [10 pg/ml (1.6 negative, and also in sepsis-like conditions such as severe fmol/ml)] detects CTpr in healthy nonsmoking persons [the burns, pancreatitis, pneumonitis, inhalational injury, bacte- upper limit of normal is 76 pg/ml (12 fmol/ml)]. There are rial meningitis, heat stroke, and severe mechanical trauma two two-site rapid assays. In Europe, a commercially avail- after extensive surgery, and also in some infections of non- able assay (LUMItest PCT, B.R.A.H.M.S. Diagnostica GmbH, bacterial causation (e.g. severe malaria or systemic fungal Henningsdorf/Berlin, Germany), shortly to be available in infections) (100–108). This phenomenon also occurs in pa- the United States, measures both ProCT and the conjoined tients with a prior thyroidectomy (100, 109). CT-CCP-I by means of a luminometer (100). This assay, Molecular sizing by gel filtration and HPLC studies in which has been used in many clinical studies, is inaccurate sepsis and in the above septic-like conditions have demon- at levels less than 300 pg/ml (24 fmol/ml). This company strated that serum components corresponding to ProCT, manufactures another more sensitive double-antibody kit, NProCT, CT-CCP-I, and the CCP-I peptide are all elevated which can reliably detect levels as low as 20 pg/ml (1.6 to varying degrees (30) (Fig. 1). The individual components fmol/ml) (82). The sensitive assays, including a recently de- were quantified with antisera specific for CT(1–32), NProCT, veloped tracer technology Kryptor assay (B.R.A.H.M.S.), the CCP-I, and also a two-site assay that used antibodies specific latter of which is commercially available in Europe, are es- to the CT and CCP-I regions of ProCT. Using capillary zone pecially useful for the detection of early forms of infection electrophoresis spectrometry and Edman sequence analysis, and for follow-up determinations (112). the serum ProCT in sepsis has been reported to lack the first two amino acids of the molecule (Ala-Pro), consequent to an Ubiquity of expression of the calcitonin-I gene in response to sepsis. aminoterminal truncation by the dipeptidyl peptidase IV Normally, immunoreactive CT is found predominantly in enzyme (EC3.4.14.5) (110). In another study, immunoreactive some neuroendocrine cells such as the thyroid C cells and ProCT and its cleavage products were extracted from pooled PNE cells, and CGRP is found predominantly in brain and septic serum using octadecylsilyl columns and characterized neurologic tissues. However, immunoreactive CT is found in by HPLC and Western blot analysis of electrophoresis-sizing many tissues throughout the body (113). Furthermore, low gels. The peptides were identified using antisera specific for levels of CT gene mRNAs have been reported to occur in liver CT, NProCT, and CCP-I (111). These investigators found (55) and also several other tissues (114). In septic hamsters, ProCT and multiple fragments of ProCT that appeared to substantially increased levels of immunoreactive CT (appar- differ in molecular size from those found in the serum of ently mostly ProCT as shown by gel filtration chromatography patients with MTC. Thus, whereas there is some uncertainty and HPLC) were found in liver, lung, kidney, pancreas, brain, as to the absolute identities of some of the fragments of ProCT heart, and small intestine (115). In this investigation, quantita-

FIG. 2. Receiver operating-curve analysis of serum CTpr for the diagnosis of sepsis in an intensive care unit, as compared with values for circulating C-reactive protein, IL-6, lactate, and pentraxin-3 (PTX-3). The sensitivity of CTpr for the diagnosis of sepsis was 89%, specificity 94%, negative predictive value 90%, and positive predictive value 94% (as assessed by the commercially available LUMItest PCT). However, the other non-CTpr markers were considerably less sensitive and less specific and had relatively poor negative or positive predictive values (modified from Ref. 102).

Downloaded from jcem.endojournals.org by on October 15, 2007 1518 J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 Becker et al. • Clincal Review tive analysis of CT mRNA expression was determined by the nating from this superfamily of genes: one of the genes yields CT/␤-actin ratio of the septic tissues in relation to the CT/␤- a very slightly different form of CGRP (CGRP-II), another actin ratio of the respective control tissues (Taq-Man technol- gene gives rise to amylin, and another gives rise to ad- ogy). Among the tissues studied, the relative increase of CT renomedullin. Furthermore, there is a gene that gives rise to mRNA, compared with controls, was, in descending order, a CT receptor-stimulating peptide with some homology to adrenal, spleen, spinal cord, brain, liver, pancreas, colon, lung, CGRP (31, 124, 125). fat, testes, and stomach (115). This phenomenon of marked Similar to the case for CT-mRNA, in septic hamster tissues increase of CT mRNA in extrathyroidal tissues also occurs in the but not in similar healthy control tissues, there also is a septic human (116). Of course, when evaluating the impact of tissue-wide expression of CGRP mRNA (126). Here, as well, each increase, consideration must be given to the total weight CGRP mRNAs are more specifically up-regulated than are of each organ. Thus, for example, the demonstration of CT the mRNAs of classical cytokines (e.g. IL-6 and TNF␣). A mRNA in fat of septic individuals assumes marked pathophys- similar phenomenon occurs for adrenomedullin. In contrast, iological significance when one considers the great bulk of amylin mRNA has not been found to be up-regulated in adipose tissue (116). nonneuroendocrine septic tissues. Thus, in sepsis several The extraordinary tissue-wide expression and secretion members of the CALC gene superfamily escape from their explains the enormous elevation of CTpr in the sera of septic normal tissue-selective expression pattern. In studies in sep- patients. The substantial suppression of CALC-I gene ex- tic humans, serum levels of CGRP and adrenomedullin are pression by nonneuroendocrine cells that occurs in normal increased in sepsis, although to levels considerably below persons is altered in septic patients by a unique stimulus those found for CTpr. arising from the infectious and/or cytokine insult that then influences the transcriptional regulation of the gene. Indeed, ProCT as a toxic factor in severe inflammation, systemic in sepsis the mRNA is more uniformly up-regulated than are infection, and sepsis the mRNAs of the classical sepsis-related cytokines, TNF␣, IL-1␤, and IL-6. In a broader sense, in sepsis the entire body Development of animal models of sepsis and correlation of serum becomes a CTpr-producing endocrine gland. It is because CTpr with sepsis and mortality. In humans, the concentration this novel form of secretion is intrinsic to the host response of serum CTpr often reflects the severity of sepsis and may to sepsis, and is reminiscent of the expression of the classical be predictive of mortality. Accordingly, to study this phe- cytokines in this condition, that CTpr are referred to as hor- nomenon more extensively, a model for sepsis was devel- mokines (115). oped in hamsters and pigs (127–131). The lack of a significant increase of CT(1–32) in sepsis merits further investigation. In part, this is indicative of a Sepsis in the hamster shift away from the normal, regulated, neuroendocrine path- Severe peritonitis was induced in hamsters by the ip im- way (characterized by a progressive posttranslational pro- plantation of pellets containing measured quantities of Esch- cessing, maturation, and secretion via secretory granules) to erichia coli, and serum CTpr was determined at intervals. a constitutive pathway (characterized by a nonstoring, bulk- According to the dosage of bacteria, the 72-h mortality in- flow secretion from nonneuroendocrine cells); this latter creased proportionately from zero to approximately 20, 70, pathway is deficient in the enzymatic processing required to and 100%. Serum CTpr also demonstrated a dose-related produce CT(1–32) (31, 117). Furthermore, once secreted, increase; at the highest dose of bacteria, serum CTpr levels ProCT and NProCT are extremely resistant to enzymatic exceeded control values by nearly 200-fold. Thus, CTpr levels degradation; in contrast, CT(1–32) is extremely labile. Thus, correlated both with the severity of bacterial insult and the circulating CT(1–32) may not be increased in sepsis because mortality. Gel filtration and HPLC studies revealed that most of its rapid degradation. Yet another factor may be the in- of the CTpr in these septic animals was in the form of ProCT fluence of heat shock proteins. These stabilizers of cellular (127). function are synthesized in response to heat, other stressful stimuli, and sepsis. In this latter illness, they may serve a Relationship between serum CTpr and cytokines. Using this an- protective role (118). Heat shock proteins also are found imal model of sepsis, the relationship of CTpr to the proximal in the blood as well as intracellularly (119, 120). They bind proinflammatory mediators, IL-l␤ and TNF␣, was studied to CT(1–32) (121), and perhaps this further augments the (128). Whereas serum CTpr remained extremely high disposal of CT(1–32) or interferes with its immunologic throughout the 24 h of this study, the increases of these detection. cytokines in the serum were less than 2-fold greater than the baseline and, importantly, were transient in duration. In Other members of the CT-gene family of peptides in sepsis: CGRP healthy hamsters, the iv administration of human ProCT and adrenomedullin. There are five genes in the CT-gene fam- caused no evident adverse effects and no changes in serum ily of peptides. The gene that was initially discovered gives IL-1␤ and TNF␣ levels. In septic animals, the ProCT injec- rise to two alternative splice variants: CT mRNA, resulting tions, albeit markedly increasing mortality (see below), only in ProCT and its components, and CGRP mRNA, giving rise modestly blunted IL-1␤ levels and did not affect TNF␣ val- to CGRP-I (122, 123). In the healthy, noninfected state, there ues. Interestingly, however, when TNF␣ was injected into is a preferential synthesis of either CT(1–32) mRNA or healthy animals, there was a 25-fold increase of CTpr levels, CGRP-I mRNA according to the cellular phenotype. Addi- compared with noninjected controls. Thus, as is the case in tionally, there are other structurally related peptides origi- the human, the magnitude and duration of the CTpr eleva-

Downloaded from jcem.endojournals.org by on October 15, 2007 Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1519 tion demonstrated its utility as a marker of sepsis in the hour of the experiment (131). In contrast to the parameters hamster. It also revealed that ProCT does not secondarily for control septic animals, all of which progressively wors- enhance levels of IL-1␤ or TNF␣ in the systemic blood. In ened and ultimately died, most physiologic and biochemical contrast, it showed that in healthy animals, TNF␣ can induce parameters of the treated animals improved or stabilized a sepsis-like elevation of serum CTpr. after infusion of the ProCT-reactive IgG (Fig. 3). Although all untreated animals had died during the experiment, most of ProCT as a toxic factor. Based on these clinical and animal the treated pigs survived until being killed (P ϭ 0.010). Thus, studies, it was hypothesized that ProCT per se may be a in this model, the findings indicate that the immunoneutral- toxic factor in sepsis and may adversely influence survival. ization of ProCT was useful in a clinically relevant situation The iv administration of human ProCT, which appeared in which sepsis was fully established and far advanced. not to be overtly injurious to normal hamsters, doubled the mortality of septic animals (129). In contrast, administration of human CT(1–32) to septic hamsters was without effect. Furthermore, a goat antiserum raised to this hormone, and shown to be completely cross-reactive with ProCT, was found to increase survival whether administered prophylactically or therapeutically.

Sepsis in the pig To investigate in detail the physiologic and metabolic con- sequences of sepsis and evaluate how ProCT immunoneutralization might affect these parameters, a larger animal model of a rapidly fatal porcine polymicrobial peritonitis was then developed (130, 131). The subsequent sepsis is similar pathophysiologically to that encountered in human disease but far more lethal. Early immunoneutralization. After having determined the structure of porcine ProCT, and, in rabbits, having produced an antiserum that was specific to the NProCT portion of this peptide, sepsis was induced in Yorkshire pigs by the ip instillation of a suspension of cecal content (l g/kg) plus E. coli (2 ϫ 1011 cfu). Simultaneous to the induction of the peritonitis, experimental pigs received an iv infusion of the ProCT-reactive purified rabbit IgG, whereas control animals received non-ProCT-reactive IgG. All animals had physiologic data (e.g. urine output, core temperature, arterial pressure, heart rate, cardiac index, and stroke index), and metabolic data (e.g. blood urea nitrogen, serum creatinine, arterial lactate, and pH) collected or recorded hourly until death or being killed (15 h after ip instillation) (130). Similarly to what occurs in humans, in this large animal model of lethal peritonitis, serum CTpr levels were found to be significantly elevated. Most of the untreated animals died within 9 h, and none survived for the 15-h duration of the experiment. However, immune IgG administration resulted in a significant improvement or a beneficial trend in most of the measured physiologic and metabolic derangements induced by sepsis. Moreover, most of these treated animals FIG.3.Top, Time course in septic pigs showing the changes in mean survived until the time of being killed, in contrast to animals arterial pressure (MAP). Time point 0 h represents commencement of treated with the non-ProCT-reactive IgG (P ϭ 0.007). the fourth hour after induction of sepsis and is the time of infusion of purified nonreactive rabbit antibody to the control group and purified Immunoneutralization of moribund pigs. In this septic pig antiporcine ProCT rabbit IgG to the treated group. After time point 0, there is a sharp fall in MAP in the control group, but the MAP of model, the physiologic and metabolic parameters of the un- the treated group remains near normal levels. *, Statistically signif- treated animals worsened rapidly, so that the animals were icant data points (P Ͻ 0.017; mean Ϯ SEM). Bottom, Time course essentially moribund by 4 h after the induction of the peri- showing the changes in serum creatinine. The graph shows very tonitis. This state is comparable with the syndrome of mul- similar creatinine concentrations before antibody infusion, which is tiple organ failure that occurs in humans with preterminal followed by a sharp rise in the control group but with concentrations that remain near normal for the treated group. Other physiologic or sepsis. Accordingly, to determine whether these gravely ill metabolic parameters also showed benefits. *, Statistically significant animals might be rescued, an evaluation of iv therapeutic data points (mean Ϯ SEM, P Ͻ 0.037), which were noted at 2, 4, and immunoneutralization was undertaken during the fourth 6 h, after which all control animals died (from Ref. 131).

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Mechanism of toxicity of ProCT in sepsis: hypotheses and early cAMP response to relatively small amounts of CT(1–32) unanswered questions has been shown to be blocked by other hormones [e.g. PTH Currently the physiologic actions of ProCT are relatively (147), epinephrine (148)]. unexplored, and it is not known how this polypeptide or its A principal function of the monocyte is its migration to components might worsen the septic process. Although sites of inflammation, a phenomenon that has been demon- acute studies have not been done in the human, the experi- strated to be induced by the addition of CT(1–32); in contrast, ence in noninfected patients with MTC indicates that chron- when patients receive therapeutic doses, this motion is di- ically high levels of ProCT do not appear to cause any ob- minished, strongly suggesting down-regulation of CT(1–32) vious ill effects. Nonetheless, although administration of receptors (149). ProCT to healthy, noninfected hamsters had no apparent ill The immunologic relevance of the monocyte response to effects, its administration to hamsters that were septic sig- CT(1–32) remains to be fully elucidated. In vivo immunologic nificantly increased mortality. This indicates that ProCT is activities of this hormone have been demonstrated in several not an initial toxic factor but requires the prior presence of studies (150, 151), and salmon CT(1–32) has been reported to a proinflammatory stimulus followed by an altered cytokine diminish the local inflammation after various forms of in- milieu. jury in rats (152). In humans with rheumatoid arthritis, eel CT(1–32) decreased production of IgG immunoglobulin Initial studies of actions of CTpr. In addition to the osteoclast, and inhibited IL-1␤ (153). However, as mentioned above, it is well known that receptors for CT(1–32) are present in CT(1–32) is not appreciably elevated in patients with severe different cell types, and multiple experimental studies of this inflammation or sepsis, and because of the marked increase peptide have been performed. However, in the past, the of serum ProCT and its component CTpr in such patients in effects of the various CTpr have very rarely been evaluated. the serum, more interest is being focused on the effects of Insofar as ProCT is concerned, one study revealed the pres- these latter peptides on the monocyte. ence of receptors to this prohormone in newborn rat calvarial cells (132). Also, the human NProCT peptide portion of CTpr and the monocyte ProCT has been reported to be mitogenic to human osteo- In vitro studies of isolated human monocytes have dem- blastic cells (U-2 OS osteosarcoma) and to induce an increase onstrated that not only CT(1–32) but also ProCT and the free in intracellular cAMP (133), although a subsequent study CCP-I peptide act as chemoattractants, inducing migration, was not confirmatory (134). In a preliminary and uncon- which is dosage dependent. This phenomenon is accompa- firmed report, a large-molecular-weight species of immuno- nied by an intracellular elevation of cAMP levels. Further- reactive CT, corresponding to an undetermined portion of more, paradoxically, these peptides may deactivate the mi- the ProCT molecule, appeared to suppress prostaglandin- gratory effects of other unrelated chemoattractants and/or E -stimulated osteoclastic bone resorption (135). It is because 2 modify monocyte surface signals (154, 155). Monocytes and of the recent awareness of the phenomenon of greatly in- neutrophils are stimulated by LPS and another proinflam- creased serum levels of CTpr in severe inflammation and matory product of bacteria, formyl methionyl leucyl phe- sepsis that studies of the action of CTpr are now underway nylalanine peptide, which induces these cells to produce an by several groups. important integrin, CD11b, a substance that is involved in The monocyte. Although the cell that has been most associated chemotaxis. Both NProCT and CGRP decrease this cellular with the action of CT is the osteoclast, it is well known that CD11b production (156). In an initial study (157), human this cell is produced from a precursor monocyte/macro- ProCT was added to mononuclear cells of peripheral blood phage cell line that also gives rise to the mature monocyte harvested from normal humans, and cytokine secretion was (136–139). Moreover, monocytes influence osteoclast activity measured in the ambient culture media. When compared and also directly induce bone resorption (139–141). Impor- with background cytokine synthesis by unstimulated cells, tantly, the monocyte plays an essential role in phagocytosis, IL-1␤ secretion was augmented 4-fold, TNF␣ 2-fold, and that T lymphocyte immune activity, and inflammation; thus, it is of IL-8 2-fold. These preliminary findings suggest that ProCT greatly involved in the initiation and course of sepsis. The might stimulate cytokine secretion from monocytes in local awareness that monocytes have CT receptors (142, 143) circulatory pools. As mentioned, TNF␣ is a known stimulus has led to several experiments investigating the effects of to ProCT secretion, and in sepsis this cytokine might locally CT(1–32) and CTpr on these cells. induce a yet further local production of this prohormone in a positive-feedback manner. The clinical impact of all of these CT(1–32) and the monocyte actions and complex interactions on the monocyte awaits clarification. Insofar as CT(1–32) is concerned, after exposure of the lymphocyte/monocyte/macrophage family to this peptide, CTpr and nitric oxide (NO). The production of the vasodilator, cAMP increases (144). This cAMP response is inhibited after NO, is elevated in sepsis (158), and this agent has been exposure of these cells to various mechanical or hormonal proposed as a mediator of the shock that may occur during stimuli (145). Also, analogous to the finding of down-regu- the course of this illness (159). However, others have relation of the osteoclast CT(1–32) receptor that occurs when ported that NO may have a beneficial role. When ProCT is hypercalcemic patients receive CT(1–32) therapy (146), a sim- added to cultures of vascular smooth muscle cells of normal ilar response to an excess of this hormone occurs in vitro rats that had previously been exposed to LPS, TNF␣, and when the monocyte CT(1–32) receptor is studied (143). The interferon-␥, the prohormone amplified the expression of the

Downloaded from jcem.endojournals.org by on October 15, 2007 Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1521 inducible NO synthase gene as well as NO production (160). teins), thus perhaps modulating the action of the CT gene The potentially detrimental effects of such an occurrence in products according to ambient circumstances (176–180). the septic process requires further evaluation. Conceivably, the much higher serum levels of CTpr may interfere with receptors or with receptor-activity-modifying CTpr and hypocalcemia. Hypocalcemia, involving particularly protein expression. Such an occurrence may block CGRP but not exclusively the ionized component, is a frequent and/or andrenomedullin activity and hence impede their concomitant of critical illness and sepsis (161, 162). Studies otherwise beneficial effects. Nevertheless, both CGRP and in the septic rat indicate that this hypocalcemia is accompa- adrenomedullin have vasodilatory actions, and whether nied by an increase of intracellular calcium (163), and a such effects may be beneficial (e.g. by increasing the blood similar intracellular increase occurs in the septic human supply to vital organs) or harmful (e.g. by inducing systemic (164). Interestingly, it is known that calcium infusions may hypotension) requires additional clarification. be harmful to septic humans (165, 166), and when septic Clearly, it is essential to further investigate the means by hypocalcemic rats are administered calcium, the fatalities which ProCT exerts its toxicity in sepsis. ProCT and its com- increase markedly (167). In a large study of critically ill pa- ponents may exert actions that differ according to their target tients, dose-related correlations were noted among the se- tissues as well as having actions that differ according to the verity of illness, the degree of hypocalcemia, and the level of ambient milieu of the host. Multiple in vitro and in vivo serum CTpr (161). Also, in this condition, serum ionized investigations will be required. calcium is known to correlate inversely with levels of TNF␣ and IL-6 (162). The cause of the hypocalcemia is unknown. CT(1–32) has been demonstrated to augment the intracellular Potential advantages of ProCT as a target for calcium concentration in several different cell lines (61, 168– immunoneutralization in the human 170). However, CT(1–32) is rarely appreciably elevated in The administration of endotoxin results in a form of sys- sepsis. A direct effect of other CTpr on intracellular calcium temic inflammation that is associated with cellular activation has not been demonstrated. In a study in hamsters, admin- and the release of inflammatory mediators (Table 2); albeit istration of human ProCT did not cause hypocalcemia; in- being different from sepsis, it is an experimental model of the deed, this prohormone was found to completely block the mediators and the control mechanisms that are relevant to hypocalcemia normally induced by injection of CT(1–32) this devastating state. In humans, it was shown that endo- (171). Thus, further studies are required to define any rela- toxin caused a rise in serum CTpr that persisted for the 24 h tionships between the CTpr peptides and serum calcium. of the experiment, although the duration of this elevation Influence of other peptides of the CT gene family. Lastly, as dis- was unknown (179). Therefore, an investigation was under- cussed above, both CGRP and adrenomedullin are increased taken to evaluate the duration of the CTpr elevation (180). in sepsis. These hormones have been reported to exert an- Serum CTpr were observed to increase by 3 h and attained tiinflammatory effects (172–175), and these actions could po- peak values at 24 h. Subsequently values very slowly and tentially be beneficial in severe infections or sepsis (e.g. an- progressively declined. Surprisingly, at 7 d, all volunteers tibacterial, increased dilatation of coronary arteries, positive still exhibited levels that were above normal (Fig. 4). In two cardiac inotropic and chronotropic effects). Furthermore, the of the subjects who were studied for a longer period, the biologic effects of the members of the CT gene family of levels did not normalize until 10–14 d. In contrast, when peptides are exerted via the same family of receptors. The subjects were administered identical doses of endotoxin and physiological profiles of these receptors are modified by cytokines were measured for 24 h, serum levels of the proin- certain accessory proteins (receptor-activity-modifying pro- flammatory cytokine TNF␣ increased at 1 h, reached a peak

FIG. 4. Exposure of human volunteers to one injection of endotoxin illustrates the differences in the release and subsequent decrease of several humoral markers of critical illness: TNF␣, IL-1 receptor antagonist (IL-1ra), IL-6, C-reactive protein (CRP), and CTpr (from Ref. 183).

Downloaded from jcem.endojournals.org by on October 15, 2007 1522 J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 Becker et al. • Clincal Review at 1.5 h, and had normalized by 24 h. Similar patterns of suggest that this prohormone may possibly be a useful target secretion occurred for the IL-1 receptor antagonist (IL-1ra), for therapeutic immunoneutralization in the human. IL-6, and granulocyte colony stimulating factor and also several other cytokines, some of which were even more transient Acknowledgments (181, 182). Thus, these short-lived acute phase cytokine elevations contrast with the extremely prolonged elevation of Received September 16, 2002. Accepted December 24, 2003. serum CTpr after a systemic inflammatory episode in healthy Address all correspondence and requests for reprints to: Dr. Kenneth humans; this suggests that CTpr not only have advantages L. Becker, Director of Endocrinology, Veterans Affairs Medical Center, as excellent markers of sepsis but also may offer a durable 50 Irving Street NW, Washington, D.C. 20422. E-mail: [email protected]. target for therapeutic immunoneutralization, even several days after the severe inflammatory illness has commenced. References 1. Copp DH, Davidson AG 1961 Evidence for a new parathyroid hormone which lowers blood calcium. Proc Soc Biol Med 107:342–344 Conclusions 2. Hirsch PF, Gauthier GG, Munson PL 1963 Thyroid hypocalcemic principle and recurrent laryngeal nerve injury as factors affecting the response to CT(1–32) was the initial peptide of the CT gene-related parathyroidectomy in rats. Endocrinology 73:244–251 family to have been isolated from MTC and normal thyroid 3. Nonidez JF 1932 The origin of the ‘parafollicular’ cell, a second epithelial component of the thyroid gland in the dog. Am J Anat 49:479 tissue, and experimental studies demonstrated its hypocal- 4. 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