British Journal of Dermatology (1976) 95, 487.

Granuloma annulare: direct immunofluorescence study PABLO UMBERT* AND R.K.WINKELMANN The Mayo Clinic and Mayo Foutidation, Rochester, Minnesota 55901, U.S.A. Accepted for publication 9 April 1976

SUMMARY

Direct immunofluorescence studies were carried out in eleven specitnens of granuloma annulare. The results (in all cases) indicate the presence of focal deposition of fibrin, localized primarily in the intcrvascular portion of the dermis, corresponding to the granulomatous and necrobiotic areas. These fitidings implicate the cellular mechatiism of delayed hypersensitivity with focal involvement of the clotting system in the development of granuloma armulare lesion.

Granuloma annulare is a benign, chronic skin disease of unknown cause characterized clinically by dermal papules and nodules, usually in annular configuration. Microscopically, the disease presents foci of necrobiotic collagen and mucinosis surrounded by mononuclear cells, often in a pallisade arrangement (Civatte, 1967). In most cases (Umbert & Winkelmann, unpublished data), histological study reveals a characteristic infiltration of the dermal collagen by mononuclear cells. Occasionally, a more granulomatous reaction is seen with an epithelioid and giant-cell component simulating the histological features of sarcoidosis (Umbert & Winkelmann, unpublished data). Although the cause of granuloma annulare is unknown, a number of possibilities have been suggested: insect bites (Moyer, 1964), sunlight (Tolmach, 1961; Dicken, Carrington & Winkelmann, 1969; Leppard & Black, 1972), diabetes mellitus (Hammond, Dyess & Castro, 1972; Haim etal., 1973), and after tuberculin testing (Beer & Wilson Jones, 1966). Whereas in the early portion of this century granuloma annulare was considered to be a tuberculid, this relationship could not be substantiated and has been discarded. We believe that granuloma annulare occurs through an immune reaction to unknown antigens. Therefore, we undertook an immunological study of granuloma annulare, and this report shovys our findings in direct immunofiuorescence studies of eleven specimens of granuloma annulare, three of necrobiosis lipoidica, and one of cutaneous sarcoidosis. The results indicate that massive, focal fibrin deposition is consistently found in granuloma annulare and suggest a cellular mechanism of delayed hypersensitivity with focal involvement of the clotting system. * Research Fellow from the University of Barcelona, Spain. Reptint requests to: Dr P. Umbert, c/o Section of Publications, Mayo Clinic, 200 First Street SW, Rochester, MN 55901, U.S.A. 487

488

P. Umbert and R.K. Winkelmann METHOD

Patient population. All eleven specimens from ten patients had classical granuloma aimulare, clinically and histologically. The biopsy specimens for immunofluorescence (IF) study were taken from the border of the lesion where there was more clinical activity. Specimen. A punch biopsy (3 mm) was done in each case, and the specimen was frozen immediately in liquid nitrogen and stored at — 70 C until used. Antisi'.ra. Monospecific antisera IgG were prepared, assayed, and conjugated with fiuorescein isothiocyanate according to the method of Triftshauser, Hayden & Beutner (1970). Monospecific antisera to human IgA, IgM, PiclIhA globulins, and fibrin were purchased from a commercial source* and tested for specificity by both immunodifiTusion (Ouchterlony) and immunoelectrophoresis. Ratios of fiuorescein to protein, units of antiserum and antibody protein assays, and dilutions at which these conjugated antisera were used conformed to previously determined standards (Triftshauser et al., 1970; Jordon, Triftshauser & Schroeter, 1971). Immunofluorescence procedures. Direct IF staining of skin biopsy specimens, performed by methods previously outlined (Jordon et al.^ I97i)> was carried out using monospecific conjugated antisera to IgG, IgM, IgA, C3, )5ic/j9iA globulins, and fibrin. Dilutions used for these antisera varied (Table i). TABLE I. Characteristics of labeled antihuman antisera Antiserum

F/P ratio*

Unitaget

Use dilution

Anti-IgG Anti-IgA Anti-IgM Anti-C3 Anti-fibrin

3-2 2-5 30 3'0 2-2

8 8 4

1:32 1:32 1:32 1:20 1:16

4 —

* Ratio of molar fiuorescein to protein. + Units determined in gel diffusion by standard methods (Beutner, Chorzelski & Jordon, 1970). RESULTS

The results in granuloma annulare (Table 2) showed the consistent presence of a fibrin mass in all eleven specimens. The mass was primarily localized in the extravascular portion of the dermis, which corresponds to the granulomatous and necrobiotic areas (Fig. i). A major feature was the absence of the fibrin mass in blood vessels; only one specimen showed a vascular deposition of the fibrin mass. Four of the eleven specimens had IgM cytoid bodies in the papillary dermis. IgM and C3 were found in the vessels of one patient. Basement membrane immunofluorescence was noted in three patients: IgM in two and C3 in the other. All three had a very fine granular pattern of deposition. In two padents, we observed IgM deposition in the dermis which had the same morphological appearance as that found for fibrin. We found C3 in three other patients; however, the amount and intensity of the fluorescence was less. In our one case of cutaneous sarcoidosis (Table 3), we found the same pattern of circumscribed deposition of fibrin mass between collagen fibers corresponding to the site of the non-caseating granuloma. * Hyland Division, Travenol Laboratories, Costa Mesa, CA 92626.

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489

TABLE 2. Results of direct immunofluorescence in ten cases of cutaneous granuloma annulare

Case I 2

3a b 4

5 6

7 8 9 10

Dermal-epidermal junction

Cytoid-like bodies

Neg. Neg. Neg. IgM, line granular IgM, fine granular Neg. C3, fine granular Neg. Neg. Neg. Neg.

Neg. Neg.

Superficial vessels

Dermis

IgM IgM

Neg. Fibrin Neg. Neg.

Fibrin Fibrin Fibrin Fibrin, IgM

IgM

IgM, C3

Fibrin, C3

IgM

Neg.

Neg. Neg.

Fibrin Fibrin, C3

Neg. Neg. Neg. Neg.

Neg. Neg. Neg. Neg.

Fibrin, C3 Fibrin Fibrin Fibrin, IgM

FIGURE I. Direct IF staining of skin lesions from patient with granuloma annulare. Positive staining with conjugate anti-fibrin ( x 80). Localized primarily in extravascular portion of dermis.

Among the three cases of necrobiosis lipoidica, two had deposition of fibrin mass in the dermal vessels and one had a weak deposition of fibrin in the dermis. DISCUSSION

Histologically, granuloma atmulare is characterized as a histio-monocytic granuloma associated with areas of necrobiosis (Civatte, 1967). Rheumatoid nodule has a similar histological appearance, with deep localization in the dermis and subcutaneous tissue and more pronounced, confluent necrobiosis with fibrinoid change. A previous review of granuloma annulare (Umbert & Winkelmann, unpublished

P.Umbert and R.K.Winkelmann

490

TABLE 3. Results of direct immunofluorescence in cutaneous sarcoidosis, necrobiosis lipoidica, and normal controls

Diagnosis Cutaneous sarcoidosis Necrobiosis lipoidica

Case

Cytoid-like bodies

Superficial vessels

Dennis

IgM, fine granular Neg.

Neg.

Neg.

Fibrin

Fibrin

Fibrin

Neg.

4

Neg. C3, fibrin. fine granular

Neg. Neg.

Neg. Fibrin, C3

Neg. Fibrin



Neg.

Neg.

Neg.

Neg.

I 2

3

Normal controls (10 cases)

Dermal-epidermal junction

data) revealed well-developed granulomatous and pallisading structures or even an 'allergic' granuloma. This spectrum of granuloma is well documented as being characteristic of several human hypersensitivity diseases. In granuloma annulare, perivascular cuffing of mononuclear infiltrate is often found (Umbert & Winkelmann, unpublished data), thus pointing to the possibility of a cellmediated immunity mechanism. In addition, we observed polymorphonuclear infiltrates only in areas that were already necrobiotic. The major findings in previous histochemical studies of granuloma annulare have been corroborated by our ::ecent histochemistry and electron microscopy studies (Umbert & Winkelmann, unpublished data), which demonstrated the presence of lymphoblast-like lymphocytes and activated macrophages associated with connective tissue changes in the dermis. The mononuclear phagocyte is a multipotential cell (Page, Davies & Allison, 1974), and its activation is the central event in a chronic inflammatory and immunopathological process such as granuloma annulare. Histochemical study demonstrates acid phosphatase and other lysosomal enzymes associated with the granuloma (Umbert & Winkelmann, unpublished data). The cause ofthe stimulus (or stimuli) that activates the mononuclear cells is unknown. The mononuclear phagocyte has the capacity to respond to different stimuli; antigenic substances (Bloom & Bennett, 1966), endotoxin (Wilton, Rosenstreich & Oppenheim, 1975), double-strand RNA, or lymphocyte-derived factors (Poulter & Turk, 1975). Recently, collagenolytic activity has been demonstrated in homogenates of rheumatoid nodules, which are capable of clearing acid-soluble tropocoUagen at neutral pH (Dabbous et al., 1974). Activated macrophages secrete collagenase (Wahl et al., 1975; Werb &: Gordon, 1975b), elastase (Werb & Gordon, 1975a), and plasminogen (Unkeless, Gordon & Reich, [974) and generate other inflammatory mediators (Page et al., 1974) such as Hageman factor, which is the initial step for the activation of complement, blood coagulation, and the fibrinolytic system (Schubert & Hamerman, 1968). The data suggest that a multi-enzyme system might be involved in the necrobiosis of nodules. Direct IF techniques provide an easy way to detect antigen-antibody complexes and complement in the tissues. Onr studies of granuloma annulare do not show immune complex disease or significant vasculitis as in the reactions associated with immediate hypersensitivity. The rare presence of immunoglobulins in the dermis or vessels does not correlate with the special pathological changes or course of granuloma annulare. The principal finding of extensive fibrin deposition is consistent with recent studies demonstrating that fibrin accumulation is a prominent and consistent feature of delayed hyperst;nsitivity skin reaction, as in the Kveim reaction and allergic contact dermatitis in man (Salo

Granuloma annulare

491

& Hannuksela, 1972; Colvin et ai, 1973). This pattern differed sharply from the intraluminal thrombi and vessel wall deposits of fibrin described in antibody-mediated reactions. Furthermore, direct evidence exists for the participation of the clotting system in cell-mediated hypersensitivity and that anticoagulants inhibit delayed reactions in animals (Schwartz & Zimmerman, 1971). The presence of extensive extravascular deposits of fibrin mass is a distinctive and consistent feature of delayed hypersensitivity skin reaction in man (Colvin & Dvorak, 1975) and in sarcoidosis (Salo & Hannuksela, t972). The fibrin mass detected was not unpolymerized fibrinogcn because of its fibrillar appearance and its characteristic periodicity as seen in electron microscopy (Dvorak et ai, 1974). Recently, studies (Colvin & Dvorak, 1975) with radioactive tracers and with IF were employed to detect and quantify fibrinogen-fibrin deposition and albumin deposition in cell-mediated immunity in the guinea-pig. Classical delayed hypersensitivity reactions to old tuberculin and to the azobenzene arsonate hapten were characterized by a progressive increase in the fibrinogen and paralleled the development of induration and erythema. The retention and resulting accumulation of fibrinogen in the delayed skin reaction seem related to a local activation of the clotting system, with polymerization of fibtinogen into insoluble fibrin. In addition to increased formation of fibrin, inhibition of fibrinolytic pathways also might have a role, as suggested by the reduction in the vessel-associated plasminogen activator in other inflammatory processes (Kwaan, 1966). The simplest explanation is that extravascular fibrin formation is a necessary consequence of increased vascular permeability, with extravasation of fibrinogen and other clotting factors. Hageman factor would be expected to be activated on contact with collagen (Wilncr, Nossel & LeRoy, 1968) or perhaps with other connective tissue components. In addition, clotting could be initiated by lymphokines (Maillard, Pick & Turk, 1972). We have found lymphokines in the sera of patients with granuloma annulare (Umbert, Belcher & Winkelmann, 1976). We would hkc to explain our findings by suggesting the possibility that the cellular hypersensitivity mechanism has an important role in granuloma annulare. Most recent studies (David, 1975) have shown that in vivo activation of macrophages requires the interaction of specific T-lymphocytes with appropriate antigen, and once activated, the macrophages may exhibit some nonspecific activity. Lymphocytes participating in cellular hypersensitivity produce a wide range of soluble mediators, some of which may interact with macrophages, causing the release of factors that induce tissue damage and destruction. The recruitment of mononuclear phagocytes into the sites of the delayed hypersensitivity reaction depends, in part at least, on chemotactic factors released by sensitized lymphocytes. These cells also produce and liberate the migration inhibition factor, which helps retain the mononuclear phagocytes at the site of inflammation and stimulates their activity. Thus, products of sensitized lymphocytes can induce macrophages to release enzymes, which may cause tissue damage and degradation together with fibrin formation at sites of chronic inflammation, such as granuloma annulare, and release lymphokines, such as migration inhibition factor, into the circulation. ACKNOWLEDGMENT

The immunofluorescent studies were done in the Cutaneous Immunopathology Laboratory, Department of Dermatology, Mayo Clinic, Rochester, Minnesota. REFERENCES BEER, W.E. & WILSON JONES, E. (1966) Granuloma annulare following tuberculin Heaf tests. Transactions of the St John's Hospital Dermatological Society, 52, 68. BEUTNER, H.H., CHORZHLSKI, T.P. & JORDON, R.E. (1970) Aiitosensitization in Pemphigus and Bullous Pemphigoid, p. 94. Charles C.Thomas, Springfield, Illinois. BLOOM, B.R. & BENNETT, B. (1966) Mechanism of a reaction in vitro associated with delaycd-type hypersensitivity. Science, 153, 80. CiVATTE, J. C1967) Histopathologie Cutanee. Flammarion, Paris.

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COLVIN, R.B. & DVORAK, H.F. (1975) Role of the cloning system in cell-mediated hypersensitivity. II. Kinetics of fibrinogen/fibrin accumulation and vascular permeability changes in tuberculin and cutaneous basophil hypei^sensitivity reactions. Journal of Immunology, 114, 377. COLVIN, R.B., JOHNSON, R.A., MIHM, M.C, JR. & DVORAK, H.F. (1973) Role of the clotting system in cell-

mediated hypersensitivity. I. Fibrin deposition in delayed skin reactions in man. Journal of Experimental Medicine, 138, 686. DABBOUS, M . K H . , YAMANISHI, Y., MAEYENS, E., HASHIMOTO, K. & HARDISON, H . (1974) Collagenolytic activity in

rheumatoid nodules. I. Effect on acid-soluble tropocollagen. Acta dermato-venereologica, 54, 265. DAVID, J. (1975) The phagocytic cell in host resistance. \i\\ A Monograph of the National Institutes of Child Health and Human Development (Ed. by J.A. Bellanti and D.H.Dayton), p. 143. Raven Press, New York. DICKEN, C.H., CARRINGTON, S.G. & WINKELMANN, R.K. (1969) Generalized granuloma annulare. Archives of Dermatology, 99, 556. DVORAK, H.F., MIHM, M.C, JR., DVORAK, A.M., JOHNSON, R.A., MANSEAU, H.J., MORGAN, E . & COLVIN, R.B.

(1974) Morphology of delayed type hypersensitivity reactions in man. I. Quantitative description of the inflammatory response. Laboratory Investigation, 31, i i i . HAIM, S., FRIEDMAN-BIRNBAUM, R . , HAIM, N . , SHAFRIR, A. & RAVINA, A. (1973) Carbohydrate tolerance in

patients with granuloma annulare: study of fifty-two cases. British Journal of Dermatology, 88, 447. HAMMOND, R., DYESS, K . & CASTRO, A. (1972) Insulin production and glucose tolerance in patients with granuloma annuiare. British Journal of Dermatology, 87, 540. JORDON, R.E., TRIFTSHAUSER, C . T . & SCHROETER, A.L. (1971) Direct immunofluorescent studies of pemphigus and bullous pemphigoid. Archives of Dermatology, 103, 486. KWAAN, H . C . (1966) Tissue fibrinolytic activity studied by a histochemical method. Federation Proceedings: Federation of American Societies for Experimental Biology, 25, 52. LEPPARD, B. & BLACK, M.M. (1972) Disseminated granuloma annulare: a variant in which the lesions involve the sun-

Granuloma annulare: direct immunofluorescence study.

British Journal of Dermatology (1976) 95, 487. Granuloma annulare: direct immunofluorescence study PABLO UMBERT* AND R.K.WINKELMANN The Mayo Clinic a...
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