Evidence for a Bactericidal Defect Correctable by Cholinergic Agonist in Vitro and in Vivo

Journal of Clinical Immunology, Vol. 2, No. 3, 1982

Short Communication

Hidradenitis Suppurativa: Evidence for a Bactericidal Defect Correctable by Cholinergic Agonist In Vitro and In Vivo

PERRI A. GINDER, ~ MARILYN OUSLEY, 1,2 D. HINTHORN, ~ C. LIU, 3 and N. I. ABDOU 1,2,4

Accepted: February 15, 1982 ration and tissue destruction increase, fibrosis, scarring, and sinus tract formation ensue (1). No Hidradenitis suppurativa (HS) affects the apocrine sweat abnormalities of immunologic function have been glands, giving chronic recurrent abscesses of axillar~' and perineal areas. We report a patient who had a defect in described in this condition. polymorphonuclear leukocyte killing of bacteria associat- In previous studies on malakoplakia, a granulo- ed with low levels of intracellular cyclic GMP. This defect matous disease with a bactericidal defect due to was corrected with a chotinergic ag0nist in vitro. Treat- failure of generation of cyclic GMP, we have dem- ment of the patient with a cholinergic agonist, bethane- onstrated an in vitro bactericidal defect and its chol chloride, resulted in prolonged clinical improve- correction in vivo by cholinergic agonists (2). ment, normal bactericidal function, and normal levels of We present a case of HS in which a defective intracellular cyclic GMP. The possible mechanisms re- polymorphonuclear leukocyte bactericidal response sponsible for the bactericidal defect and for the patient's to phagocytosed Escherichia coti and staphylococci improvement are discussed. was found. The defect was associated with low KEY WORDS: Hidradenitis; cholinergic agonist; bactericidal defect. levels of intracellular cyclic GMP. These defects could be corrected in vitro by the incubation of INTRODUCTION patient's leukocytes with cholinergic agonists. Ad- ministration of the cholinergic agonist to the patient Hidradenitis suppurativa (HS) is a chronic, suppu- corrected the neutrophil killing defect and resulted rative, and cicatricial disease of apocrine gland- in healing of abscesses and sinus tracts. bearing skin areas, principally axillary and anogeni- tal skin. Tropical climate, obesity, and acne are CASE REPORT known to be predisposing factors, but the etiology is unknown. The sequence of histologic changes This 70-year-old Caucasian male was admitted to progresses from keratinous plugging of the apocrine the University of Kansas Medical Center in March duct and proliferation of bacteria trapped beneath 1980 for evaluation of persistent hidradenitis sup- the plug lead to rupture of the gland and subsequent purativa for 10 years, involving axillae and perine- extension of infection to adjacent glands. As suppu- um, with ulcerations, abscesses, and draining sinus tracts. These abscesses were not associated with tDepartment of Medicine, Division of Allergy, Clinical Immunol- constitutional symptoms. He had undergone multi- ogy, and Rheumatology,University of Kansas Medical Center, ple drainage procedures throughout the years and Kansas City, Kansas 66103. had had two attempts at marsupialization of the 2The Kansas City Veterans Administration Medical Center, Kansas City, Missouri 64128. perineal tracts in 1979. Mixed aerobes and anaer- 3Division of Infectious Diseases, University of Kansas Medical obes grew on culture, including enterococci, staph- Center, Kansas City, Kansas 66103. ylococci, E. coli, streptococci, Bacteroides melan- 4To whom correspondence should be addressed at Division of Allergy, Clinical Immunology, and Rheumatology, 4035 B, inogenicus, corynebacterium species, and Klebsiel- University of Kansas Medical Center, Kansas City, Kansas ta enterobacter. He had been given multiple 66103. courses of antimicrobial therapy including 6 weeks

237 0271-9142/82/0700-0237503.00/0 ~' 1982 Plenum Publishing Corporation 238 GINDER, OUSLEY, HINTHORN, LIU, AND ABDOU of cephalexin, 4 weeks of gentamicin, 2 weeks of Evaluation of T-Ceil and B-Cell Subpopulations moxalactam, and 3 weeks of ceforanide, each of Enumeration of T and B cells was performed by which resulted in limited short-term improvement. standard methods using the E-rosette assay for T He had no past history of recurrent sinopulmonary cells (3) and surface immunoglobulin assay for B infection, inflammatory bowel disease, connective cells (4). The in vitro proliferative response of T tissue disease, or diabetes mellitus. He did have cells to phytohemagglutinin and the in vitro moderate chronic obstructive pulmonary disease immunoglobulin biosynthesis of B cells in response and atherosclerotic cardiovascular disease with to pokeweed mitogen were performed by described chronic stable angina Pectoris and mitrat insuffi- methods (5). ciency. On examination, there was a 2 x 2-cm painful, warm fluculent mass in the left axilla. The perineum had multiple draining sinus tracts with Chemotaxis associated swelling, warmth, and tenderness. Polymorphonuclear leukocytes were prepared by Routine complete blood count, urinalysis, elec- the dextran sedimentation method (2). We per- trolytes, and liver and kidney functions were nor- formed the standard chemotaxis assay using Skyes- mal. Quantitative serum immunoglobulins were 920 Moore chambers and 5-~m-pore size Nucleopore mg/dl IgG, 390 mg/dl IgA, and 48 mgtdl IgM, all filters (Nucleopore Company, Pleasanton, CA). within normal range. Skin tests for delayed hyper- Chemotactic factor placed in the lower chamber sensitivity including PPD, streptokinase-strepto- was a 1:3 dilution of fresh human plasma activated dornase, and mumps were negative. Antinuclear with 10 mg/ml of zymosan (Sigma Company, St. antibody was negative. Serum complement (CH50) Louis, MO) or with E. co!i culture supernatant. The was found to be normal, 210 CH50 units. Deep plasma was either from our patient or from normal bioPsY specimens of perineal sinus tracts showed healthy controls. In upper chambers 3 x l0 6 leuko- acute and chronic inflammation, ulceration, and cytes were suspended in 1 ml of medium I99 (K. C. abscess formation. Acid-fast bacillus and fungal Biologicats, Lenexa, KS). The chambers were incu- stains and cultures were negative. Michaelis-Gut- bated at 37°C for 90 min. The membranes were then mann bodies, histologically specific for malakopla- fixed with methanol, air-dried, and stained with kia, were not identified and there were no granulo- 0.5% safranin. Ten fields at magnification x600 mata detected in the biopsies. were counted. In all the chemotaxis assays, we Extensive leukocyte function studies were under- recorded spontaneous migration of cells to the taken, disclosing normal phagocytosis and chemo- lower chamber in the absence of chemotactic fac- taxis, but defective bactericidal function as detailed tor. below. This defect was correctable in vitro by the addition of cholinergic agonists. Antibiotics were Phagocytosis discontinued and the patient was started on bethanechol chloride, 10 mg three times daily; this was Purified preparations of neutrophils by the dex- increased to 50 mg three times daily 1 week later. tran sedimentation method or of mononuclear cells After 1 month on bethanechol treatment there was by the Ficoll-Hypaque method were used (2). We striking improvement; pain and swelling decreased, incubated cells from the patient or from normal purulent drainage stopped, and sinus tracts began to controls with opsonized latex particles (Dow Chem- heal. The patient was continued on bethanechol ical Company, IN) for t hr at 37°C. Aliquots of the chloride and was followed for approximately 14 cell suspensions were centrifuged in cytospin cham- months without evidence of recurrence of the ab- bers (Shandon Company, Sewickley, PA), air- scesses. dried, and stained by the Wright's method. Cells that phagocytosed latex particles were counted. MATERIALS AND METHODS Serum Immunoglobulin and Complement Protein Bactericidal Assay Assays One x l06 leukocytes containing >95% neutro- These were performed by the standard quantita- phils from the patient or normal controls were tive immunoplates for the former and CH50 by the incubated with 5 x 106 E. coli or Staphylococcus standard hemolytic assay for the latter. aureus organisms for 1 hr at 37°C on a rotator. Cells

Journal of Clinical Immunology, Vol. 2, No. 3, 1982 BACTERICIDAL DEFECT IN HIDRADENITIS 239 were then washed, and 0.25% sodium deoxycholate Table I. In Vitro Killing of E. coli or S. aureus by Blood (Sigma Company) was added to lyse cell mem- Neutrophils branes. Serial dilutions of the mixture were inocu- No. of bacterial colonies (mean _+ SD) - lated onto beef broth agar. After incubation for 24 Neutrophil hr at 37°C, bacterial colonies were counted (2). source Manipulation E. coli S. aureus

Normal None 6 + 4 2 + 2 Quantification of Intracellular Cyclic Nucleotides Patient None 139 -+ 23 54 +- l l Normal Carbachol in vitro b 5 +_ 2 3 +- 2 Normal or patient's leukocytes were suspended Patient Carbachol in vitro b 3 -+ ! 17 ._-2 3 Patient Bethanechol in vivo C 7 -+ 1 l l -+ 7 in Hanks' balanced salt solution at a concentration of 1 x 107 cells/ml. The cells were preincubated aMean -+ SD of two experiments, each done in duplicate. bl0 -5 M carbachol incubated with cells for 30 min at 37°C prior to either with 10 -5 M carbachol or with Hanks' bal- performing the bactericidal assay. anced salt solution for 30 rain at 37°C. This proce- cTested 2, 4, 6, and 9 months while on 150 rag/day bethanechol dure was followed by the addition of 5 ml of chilled chloride. Cells were not treated with the drug in vitro. 6% trichloroacetic acid, and cell suspensions were sonified for 1 min with a Branson instrument (Bran- son Sonic Power Co., Plainview, NY). The tubes tactic agent. There was no difference between pa- were centrifuged at 5000g for 10 rain, and the tient's plasma and normal plasma in their capacity supernatant was removed. The tricholoroacetic to generate chemotactic factor when activated by acid was then removed by extraction with ether, zymosan. The chemotaxis assay was repeated twice and the precipitate redissolved in Tris-EDTA buff- and the values shown are the means of the experi- er. We assayed cyclic AMP and cyclic GMP by ments, each done in triplicate. competitive protein binding radioimmunoassay (6) using commercial kits (Amersham-Searle, Arling- ton Heights, IL). Phagocytosis Assay Neutrophils from the patient's blood had a phago- RESULTS cytic capacity similar to that of normal cells. The proportions of patient's or normal's neutrophils capable of phagocytosis were 91 +- 12 and 87 -+ 9% Lymphocyte Studies (mean -+ SD), respectively. These values were not Normal numbers of T and B cells were found in significantly different whether normal or patient the peripheral blood--87% T cells and 7% B cells; serum was used to opsonize the latex particles. The normal values were 82 -+ 7% for the former and 6 -+ phagocytosis assay was repeated twice and the 3 for the latter. The in vitro mitogen response of the values shown are the means of the experiments, patient's blood lymphocytes to phytohemagglutinin each done in triplicate. was poor. Specific incorporation of tritiated thymi- dine was 5. Normal controls done simultaneously Bactericidal Activity had values of 97 +_ 37. The in vitro IgG biosynthesis by patient's lymphocytes was normal; patient's Leukocytes from the HS patient had decreased culture gave 937 ng IgG/culture, and normals 863 -+ bactericidal activity. The number of E. coli colonies 305 ng IgG/culture. All the lymphocyte studies were present in the disrupted HS cells was over 20 times repeated at least three times and the values shown greater than that from normal controls (Table I). are the means of the various experiments, each This defect could be corrected in vitro by incuba- done in triplicate. tion with 10 -5 M carbachol, which decreased the number of colonies relative to that of controls, Chemotaxis Assay Leukocytes from the patient tested 2, 4, 6, and 9 months after the institution of in vivo treatment with Neutrophils from the patient had directed chemo- 150 mg bethanechol daily were as effective as taxis values similar to controls, 37 + 13 for the normal cells in killing E. coli (Table I). When the patient's ceils and 33 -+ 7 for controls. The same patient discontinued therapy for several months on conclusions were reached whether E. coli filtrate or his own initiative, suppurating lesions recurred. zymosan-activated plasma was used as the chemo- There was healing upon the reinstitution of bethane-

Journal of Clinical Immunology, Vol. 2, No. 3, 1982 240 G1NDER, OUSLEY, HINTHORN, LIU, AND ABDOU

Table II. Cyclic Nucleotide Levels in Polymorphonuclear Cells defect or absence of tubulin subunits or failure of Nucleotide levels generation of cyclic GMP (7, 8, 11). The latter Treatment (pmol~() -7 cells) enhances lysosomal degranulation by signaling mi- Cell source In vitro In vivo Cyclic AMP Cyclic GMP crotubule assembly (7). This appears to be modulat- ed by adrenergic and cholinergic agents which Normals~ None None 9.7 -+ 4.5 0.93 + 0.22 provoke changes in the concentration of cyclic Patient None None 8.4 -+ 2.1 0.20 ± 0.07 Normal Carbachol None 7.9 + 3.2 0.84 ± 0.23 nucleotides (t0). In this patient the defect might be Patient Carbachol None 8.2 --- 1.9 0.89 ± 0.11 attributed to the low level of cyclic GMP in poly- Patient None __b 6.9 ± 2.7 0.77 ± 0.07 morphonuclear leukocytes, resulting in decreased ~Three healthy laboratory personnel. lysosomal degradation and the inability of the cells bBethanechol chloride, 150 rag/day. to release the lysosomal enzymes. It is unlikely that the bactericidal defect of neutrophils seen here chol. Unfortunately, we did not have the opportuni- could explain the localization of the disease or its ty to do in vitro studies during the period off histological changes. Such a defect, however, could therapy. influence the expression of the disease. The ob- served neutrophil abnormalities in our patient were probably not secondary to HS per se, since we Cyclic Nucleotide Levels cannot demonstrate similar defects in three other The patient's leukocyte cyclic AMP levels before patients with HS (unpublished observations). or after in vitro treatment with carbachol were not One problem in our assay has been in dissecting significantly different from those of leukocytes of the kinetics of bacterial uptake by the neutrophils. three normal controls (Table II). In contrast, cyclic The increased number of colonies grown after lysis GMP was very low in the cells from the HS patient, of the patient's cells could reflect augmented phago- when compared to levels of normal cells. Cyclic cytosis rather than decreased killing. We have GMP increased to almost-normal levels after in attempted to disprove this in a control experiment. vitro treatment of the HS cells with carbachol or in An aliquot of cells that was incubated with bacteria vivo treatment of the patient with bethanechol chlo- for 1 hr was saved. These washed cells were ride (Table lI). incubated for an additional hour before lysis. The numbers of colonies grown after lysis and plating of the latter cells were compared with those plated DISCUSSION after the initial hour of incubation. We found no These studies clearly demonstrate that peripheral difference in patient colonies from the first and blood neutrophils of the patient with HS had de- second incubations, while control colonies marked- creased bactericidal activity for E. coti and staphy- ly dropped. This was interpreted as an inability of lococci and low levels of cyclic GMP. These defects patient cells to kill the phagocytosed bacteria. In were correctable in vitro and in vivo by cholinergic addition, visual inspection of the cells which phago- agonists. Correction of these defects correlated cytosed latex particles disclosed no difference be- with clinical improvement of the patient. tween patient and control neutrophils regarding the The etiology of HS is unknown. Clearly, it is not number of cells with latex particles. The possibility a primary infectious process, because of its anatom- of carry-over of bacteria after cell washing was ic limitation to apocrine gland-bearing skin. Intra- excluded by the failure to grow colonies from the cellular metabolic defects or imperfect phagolyso- wash supernatants. some formation can lead to a bactericidal abnormal- The abnormalities in our patient are similar, in ity of phagocytic cells (2, 7, 8). The intracellular some respects, to the neutrophil abnormalities in metabolic defects are numerous and could include the Chediak-Higashi syndrome (7, 8, 1I) and mono- the absence of respiratory burst, ineffective glucose cyte abnormalities in malakoplakia (2) and atypical utilization, inadequate superoxide formation, and mycobacterium infection (12). In these conditions, deficient H202 and myeloperoxidase (9). Release of decreased bactericidal activity with or without de- lysosomal enzymes from the phagolysosome re- creased lysosomal degranulation and abnormal gi- quires intact microtubules (7). Abnormalities of ant lysosomal granules have been demonstrated and phagolysosome formation may be attributed to fail- were corrected by cyclic GMP (7, 8), cholinergic ure of microtubular assembly due to structural agonists (2, 7), or ascorbate (I I, 13). It is thought

Journal of Clinical Immunology, VoL 2, No. 3, 1982 BACTERICIDAL DEFECT IN HIDRADENITIS 241

that proper microtubule assembly during phagocy- 2. Abdou NI, Napombejara C, Sagawa A, Watanabe I, Stechs- tosis is required for rapid lysosomal fusion with the chulte DJ, Lindsey N, Allen M: Malakoplakia: Evidence for monocyte lysosomal abnormality correctable by chotinergic phagocytic vacuoles in normal cells. In the Che- agonist in vitro and in vivo. N Engl J Med 297:1413-1419, diak-Higashi syndrome microtubules may fail to 1977 assemble after phagocytic stimulus, leading to a 3. Jondal M, Holm G, Wigzetl H: Surface markers on human T reduced rate of lysosomal degranulation. This fail- and B lymphocytes. I. A large population of lymphocytes ure may be due to the absence of cyclic GMP, forming non-immune rosettes with sheep red blood cells. J Exp Med 136:207-215, 1972 which acts as a signal to microtubule assembly (14), 4. Abrahamsohn I, Nilsson UR, Abdou NI: Relationship of although such abnormalities have not been seen by immunoglobulin to complement receptors of human B cells. all observers (11, 15). J Immunol 112:1931-1938, 1974 We cannot exclude the possibility that these 5, Abdou NI, Lisak RP, Zweiman B, Abrahamsohn I, Penn cellular functional defects could be an epiphenome- AS: The thymus in myasthenia gravis: evidence for altered cell populations. N Engl J Med 291:1271-1275, 1974 non of chronic inflammation and not a pathologic 6. Gilman AG: A protein binding assay for adenosine 3'5' entity. Moreover, little is known about the effect of cyclic monophosphate. Proc Natl Acad Sci USA 67:305- long-term cholinergic agonist administration on 309, 1970 apocrine gland secretions. An inhibitory effect by 7. Oliver JM, Zurier RB: Correction of characteristic abnor- the drug on glandular secretory activity resulting in malities of microtubule function and granule morphology in Chediak-Higashi syndrome with cholinergic agonists; Stud- a reduction of the nutrient milieu for infection ies in vitro and in vivo in the beige mouse. J Clin Invest cannot be dismissed. 57:1239-1247, 1976 There are unanswered questions about host-de- 8. Boxer LA, Rister M, Allen JM, Baehner RL: Improvement fense abnormalities such as those seen in this of Chediak-Higashi leukocyte function by cyclic guanosine patient with HS. Our patient was anergic and had a monophosphate. Blood 49:9-17, 1977 markedly depressed in vitro blastogenic response to 9. Babior BM: Oxygen-dependent microbial killing of phago- cytes. N Engl J Med 298:659-668, 1978 phytohemagglutinin despite normal numbers of T 10. Zurier RB, Weissermann G, Hoffstein S, Kammerman S, Tai cells. The mechanisms responsible for the low HH: Mechanisms of lysosomal enzyme release from human levels of cyclic GMP and its relationship to anergy, leukocytes. II. Effects of cAMP and cGMP, autonomic poor mitogen responses, and a phagocytic cell agonists and agents which affect microtubule function. J Ctin abnormality suggest a broad immune defect. Chron- Invest 53:297-309, 1974 11. Gatlin JI, Elin RJ, Hubert RT, Fauci AS, Kaliner MA, Wolff ic indolent infection may cause blunting of immune SM: Efficacy of ascorbic acid in Chediak-Higashi Syndrome function as described in leprosy (16). The imbalance (CHS): Studies in humans and mice. Blood 53:226-234, 1979 of intracellular levels of cyclic nucleotides could 12. Gardner JD, Ousley M, Godfrey W, Lindsey NJ, Abdou NI: result in enhanced prostaglandin E2 release. The Mycobacterium fortuitum infection. Evidence for bactericid- latter could then suppress T-cell proliferation-func- al defect due to hyperactive antigen specific suppressor tion and the bactericidal function of phagocytic cells. Correction in vitro and in vivo by cholinergic agonist and indomethacin. Am J Med (in press), 1982 cells (17). Although these features require further 13. Boxer LA, Watanabe AM, Rister M, Besch HR, Allen J, work, it is clear that cholinergic agonists were Baehner RL: Correction of leukocyte function in Chediak- helpful in the therapy of the patient reported in this Higashi syndrome by ascorbate. N Engl J Med 295:1041- paper and merit further evaluation in the treatment 1045, t976 of HS or in other cases of neutrophil killing defects. 14. Oliver JM: Impaired microtuble function correctable by cyclic GMP and cholinergic agonists in the Chediak-Higashi syndrome. Am J Pathol 85:395--4t8, 1976 ACKNOWLEDGMENTS 15. Frankel FR, Tucker RW, Bruce J, Stenberg R: Fibroblasts The work was supported by the Veterans Admin- and macrophages of mice with the Chediak-Higashi-like syndrome have microtubules and actin cables. J Cell Biol istration. Dr. C. Liu is a research career awardee of 79:401-408, t978 the National Institutes of Health. We thank Mrs. 16. Touw J, Stoner GJ, Belehu A: Effect of mycobacterium Anne Knight for secretarial assistance and Mrs. leprae on lymphocyte proliferation: suppression of mitogen Deborah Marino for technical assistance. and antigen responses of human peripheral blood mononuclear cells. Clin E×p Immunol 41:39%402, 1980 REFERENCES 17. Fischer A, Durandy A, Griscelli C: Prostaglandin E2-mediat- ed monocyte suppressive activity--Role in immunoregula- 1. Hurley HJ: Apocrine glands. In Dermatology in General tory disorders. In Primary Immunodeficiencies, M Selig- Medicine, TB Fitzpatrick (ed). New York, McGraw-Hill, mann, WH Hitzig (eds). New York, Elsevier Nerth-Holland, 1979, pp 480-483 1980, pp 363-372

Journal of Clinical Immunology, VoL 2, No. 3, 1982