Diagnostic Procedures in Immunodermatology

DIAGNOSTIC PROCEDURES IN IMMUNODERMATOLOGY

RUDI H. CORMANE, M.D., AND SYED SHAFI ASGHAR, PH.D. Department of Dermatology, Binnengasthuis, University of Amsterdam, Amsterdam, The Netherlands

Most immunologic diseases are caused by the derailment of the humoral or cellular pathways of the immunologic defense system. This derailment results from numerous factors such as the inability of the patient to remove the pathogen; the consumption, defect, or deficiency in any component of these pathways, and the overproduction of any of the components. To diagnose these immunologic disorders one has to detect the pathogen and the reactions caused by it and to determine the cause of its nonclearance. The immunofluorescence technique has been invaluable in detecting both the antigen that causes the disease and the reactions initiated by the antigen, such as the production of antibodies and the activation of the complement system. The immunoperoxidase technique has also been used for these purposes in certain instances. For detecting the circulating immune complexes which occur as intermediates in the chain of reactions initiated by the antigen, various physicochemical and biologic techniques have been used. However, none of these tests seems to be totally reliable for determining whether circulating immune complexes are present. The consumption of complement was detected by hemolytic estimations and radial immunodiffusion or rocket electrophoresis. These techniques were also useful in detecting the hereditary deficiencies in immunoglobulins and components of classical and alternative pathways of complement activation. Since these techniques cannot be used to estimate IgE, the radioallergosorbent test was used to measure such levels in atopic patients. Cellular hypersensitivity was detected with skin tests together with methods which assess the ability of lymphocytes to produce mediators in response to antigen. Many of these media tor assays, however, are not suitable for this purpose. A satisfactory substitute appears to be to determine the factor in antigen-stimulated, lymphocyte culture supernatants which acti vates macrophages to take up radiolabeled colloidal gold or radiolabeled glucosamine. In con tact allergic dermatitis, an increase in the IgD-bearing lymphocytes and granulocytes has also been correlated with cellular hypersensitivity. Lymphocytes and polymorphonuclear leuko cytes coated with antibodies mainly directed against nuclear antigens of the basal layer cells of the noninvolved epidermis have invariably been encountered in psoriasis. The use of these findings for diagnostic purposes and for understanding the mechanisms of certain diseases is being explored.

The factors that cause the various immunologic genetic abnormalities. These factors can cause diseases in man can be broadly divided into four derailment of the humoral or cellular pathways of categories: (1) self antigens; (2) foreign antigens immunologic reactions. Figure 1 shows the main such as viruses, bacteria, chemicals, and drugs humoral immunologic reactions but not the side which may form immunogens with body proteins; reactions, such as abnormalities in the mechanism (3) foreign substances such as bacterial polysac of blood coagulation caused by the activation of charides; and (4) immunodeficiencies and other alternative pathway of complement activation. In normal individuals, these reactions appear to be inhibited by agents in the blood, but they could Reprint requests to: Dr. R. H. Cormane, Department of be activated by'factors such as endotoxins or by an IJermatology, Binnengasthuis, University of Amsterdam, A. msterdam, The Netherlands. tigens if antibodies have already been produced. Abbreviations: After the source of their activation is removed, they ECF-A: eosinophil chemotactic factor of anaphylaxis return to the unactivated state. In a disease state, FTC: fluorescein thiocyanate some of these reactions are activated, depending LPF: lymphocyte proliferation factor on whether the activator is an antigen or an endo MAF: macrophage activator factor MIF: migration inhibition factor toxin, and remain so during the course of the dis MNLCTX: mononuclear chemotactic factor ease either because the activator is continuously PG: prostaglandin resupplied or because the patient's reticuloendo PMN: polymorphonuclear thelial system or some other eliminating mecha RAST: radioallergosorbent test SRS-A: slow-reacting substance of anaphylaxis nism is unable to remove it. Other causes of disease 129 130 CORMANE AND ASGHAR Vol. 67, No.1

IgE-sensitized _ circulatiflg ...----- Ag + Ab Basophil or Mast cell Activating substance such baS01PhiiS immune complex IgE (Ab) IgE (Ab) as endotoxin polysaccharide I '- .J + allergen (Ag) ~ Clumping of platelet, i IF---IF Release of chemical mediators I release of vasoacti ve I Platelet-activating amines Immune complex (IC) C3+P ,. P factor ------.. (deposited) ---,- increased permeability I ~ C4 C2 : +C3PAse (Factor D) C CI C3 Mg+ + , 1- -tV ): ______C3PA(FactorB) C42 ------l.. ,/,:r "C3b inhibitor /' ~/ C3a C3b-C3bi I ...... ------I Attraction of I PMN-leukocyte I Tissue injury ...----- chemotactic 1------C5 and release of lyso factors somal constituents _------C5aI \ C5b 1+ C6-C9 C5b- C9 membrane attack

FIG. 1. Summary of main humoral immunologic reactions in clinical hypersensitivity and disease. Ag = antigen, Ab = antibody, CI-C9 = components of complement, IF = initiating factor, P = properdin, C3PA = C3 proactivator, C3PAase = C3 proactivator convertase. states include a defective component of these path allergic disorder in atopic patients, i.e., IgE anti ways, e.g. , a deficient inhibitor of the first compo bodies which combine with mast cells through Fc nent of complement in hereditary angioneurotic fragments. When allergen reacts with cell-bound edema and the overproduction of IgE in atopic pa IgE, the enzyme sequence is activated and such tients. chemical mediators as histamine, the slow-react First, we shall describe these pathways briefly. If ing substance of anaphylaxis (8R8-A), prosta antibodies have been produced by an antigen, glandins (PGs), eosinophil chemotactic factor of addition of more antigen may lead to the formation anaphylaxis (ECF -A), and kallikrein are released. of antigen-antibody complex. This complex may The cellular pathways of self-defense involve be fixed or circulating depending on whether the various cell types. One of them, the B-Iymphocyte, antibodies have been produced against a fixed is the immunoglobulin-bearing lymphocyte found antigen such as autoantigen or whether the antigen in lymph nodes. The other cell, the T -lymphocyte, is circulating or is one of the proteins of the is the monocyte which originates in the bone circulatory system. If the complex is circulating marrow, is processed in the thymus, and is devoid and large ( > 198), it will be deposited between the of antibody. At least these two types are needed to endothelial cells in such a way [1] that a platelet express cellular hypersensitivity. When sensitized activating factor is released from IgE-sensitized lymphocytes come in contact with antigen, they basophils by immune complexes and the vasoac release several proteins, such as the migration tive amines are released by the platelet-activating inhibition factor (MIF), the skin reactive factor, factor. Because of increased vascular permeability, and about 10 more which have a pronounced effect larger complexes are trapped in and around the on other cells involved. These factors, perhaps by wall of the blood vessel. autocatalytic or positive feedback effect, help to Whatever the mechanism of deposition, the im produce biologic activities from nonsensitized mune complexes either before or after deposition lymphocytes as well. By virtue of their different can activate the complement sequence that leads properties, they lead to the localized accumula to the generation of chemotactic factors and is fol tion of nonspecific inflammatory mononuclear cells lowed by the attraction of polymorphonuclear and, in extreme reaction, to tissue necrosis (see (PMN)-leukocytes, the lysosomal enzymes of which detailed descriptions by Lawrence and Lardy may cause tissue injury [2]. [4), David [5), and Asghar and Cormane [6]) . If the alternative pathway has been activated, Both humoral and cellular immunologic path say by endotoxin, complement from the C3 stage ways may be operative at the same time in a will be activated by a mechanism described by patient or an experimental animal. Besides their Muller-Eberhard [3], which involves an initiating activation by certain pathogens, some genetic factor, properdin, C3 proactivator, C3 proactivator deficiency, defects or overproduction may eventu convertase, C3, and probably unknown proteins of ally impair the function of the whole pathway and this pathway. After complement from C3 has been may cause the disease. activated, the remaining steps of complement We have described these pathways briefly be action leading to tissue injury and membrane cause our aim is not to discuss the mechanisms of attack are common to both pathways. immunologic reactions but to show that the In addition to the mechanism that activates the method of diagnosing immunologic disorders complement system, there are other causes of should be based on either a recognition of the July 1976 IMMUNODERMATOLOGY 131 factors that are responsible for initiating the dis and bacterial antigens with rhodamine and fluores ease or the detection of reactions which have been cein thiocyanate (FTC}-labeled antisera occasion activated by these factors. Thus, the diagnosis of ally revealed them in combination. Parish [10] has immunologic diseases should be based on the also demonstrated the antibody specificity of im criteria described in Table 1. munoglobulins against the antigen in the lesion. The extracellular isolated protein antigen of Strep. DETECTION OF ANTIGEN pyogenes group A and antigen extracted from In an immunologic reaction caused by an anti Staphylococcus aureus were unequivocally fixed on gen, microprecipitation of antigen and antibodies aggregated IgG in the lesions. may occur in and around the blood vessel walls or The immunofluorescence technique is now es immune complexes may be found in the circulation tablished as a diagnostic and investigative tool in before they have been deposited. So far, there is a immunodermatology. Another technique, which scarcity of data showing antigen parts of immune uses other markers, is the immunoperoxidase tech complexes which are circulating, but in certain nique in which peroxidase-coupled antibody reacts cases the immunofluorescence technique has been with antigen and the colored enzyme product used to demonstrate the antigen in the deposited serves as a stain. Although this technique is not as immune complexes. The rationale and the techni sensitive as the immunofluorescent technique [11], cal details of the immunofluorescence technique it is useful for certain purposes in light and in have been described by Beutner et al [7], and the electron microscopy [12]. requisites for achieving peak performance with immunofluorescence microscopy have already DETECTION OF ANTIBODIES been discussed by Ploem [8]. Direct and indirect immunofluorescence tech Using this technique, Wemambu et al [9] found niques have been useful in detecting antibodies, evidence that microbacterial antigen is the cause both fixed and circulating. Several antibodies of the Arthus-like reaction in a form of leprosy under different pathologic conditions have been characterized by neutrophil infiltrates. Parish [10] detected by this technique, but only the most demonstrated Streptococcus pyogenes group A and important are listed in Table II. D antigens in cutaneous vasculitis and erythema Antibodies may also occur on the surface of cells nodosum. Streptococcal, candidal, and microbac such as lymphocytes or PMN-Ieukocytes (see terial antigens were also detected in the lesions of below). vasculitis. Simultaneous staining for the immunoglobulins DETECTION OF IMMUNE COMPLEXES The mechanism of circulating immune complex TABLE I. Basis of diagnosis various immunologic disorders disease is the same as that of fixed complexes except that active processes are involved in the 1. Detection of antigen Whether fixed, circulating, deposition of immune complexes. The persistent self or foreign. Identifica presence of complexes can cause such pathologic tion if possible. effects as anaphylaxis, Arthus-like reaction, granu 2. Detection of antibod- Whether locally fixed, cir lomata, and hyperviscosity. Immunofluorescence les culating, or on the sur microscopy is now indispensable for detecting face of ancillary cells. deposited immune complexes. The circulating 3. Detection of immune Whether circulating or complexes can be detected by various physico complexes fixed. chemical and biologic methods (Tab. III). 4. Involvement of com Whether complement is ac plement by classical tivated locally or sys Physicochemical Methods or alternate pathway temically. Whether com The physicochemical methods depend on the plement levels are altered separation of complexes from serum immunoglob and fragments detecta ulins because of size, charge, or solubility charac ble. teristics. Analytical ultracentrifugation has been 5. Immunodeficiencies Whether there is deficiency used to demonstrate 22S IgG-IgM complexes in and other genetic ab or genetic defect in im the serum of patients with rheumatoid arthritis normalities munoglobulins, comple

ment components of clas TABLE II. Antibodies detectable in the skin and/or serum sical or alternative path by immunofluorescence technique in various diseases way, or inhibitors of these systems. Substrates for antibodies Diseases Detection of cellular Whether delayed hyper Subcellular constituents SLE hypersensitivity and sensitivity is operative. Stratum corneum Psoriasis vulgaris studies of ancillary Whether antibodies di Intercellular space material Pemphigus, burns cells rected against a particu Basal cell cytoplasm a and Drug reaction lar antigen are present on membrane ancillary cell surface. Basement membrane Pemphigoid 132 CORMANE AND ASGHAR Vol. 67, No.1

TABLE III. Methods employed for the detection of work is needed to confirm the suitability of this circulating immune complexes method for immune complex detection. Immune complexes were detected by virtue of Physicochemical methods Biologic methods their property to release histamine from guinea-pig Analytical ultracentri Reaction with Clq lungs [17] and to aggregate platelets [18]. When fugation antigen is known to be in complexes, an increase in Density gradient ultra Reaction with rheumatoid free antibody concentration after enzymic degra centrifugation factor dation of antigen suggests the presence of immune Electron microscopy Platelet aggregation complexes. DNA-anti-DNA complex has been de Cryoprecipitation Histamine release from tected by this method [19]. guinea-pig lung Decreased levels of total complement or a spe Differential solubility Increased antibody con cific component of complement (C3) associated centration after de with the degradation products of complement (~lC struction of antigen - ~lA + a2D) have been used to measure circulat Anticomplementary ing immune complexes. Recent data on the alter activity native pathway, however, suggest that these obser vations implicate a nephritic factor or the activa tion of the alternative pathway at any other step and 11-13S IgG-IgG complexes in the serum of [3 ]. W aldenstrom 's benign hypergammaglobulinemic Thus, these biologic tests do not conclusively purpura [13]. This technique, however, is useful identify the immune complexes circulating at any only for detecting large quantities of circulating one time. Several factors affect the accuracy of complexes (>5% of total serum proteins). Other these tests: the degree of lattice formation required complexes can be detected by density gradient to initiate the biologic test reaction, the excess of centrifugation. Immune complexes in the sediment antigen or antibody in the sera at the time of have been visualized by electron microscopy [14]. testing, pseudoimmune complexes which behave Other methods of detecting immune complexes like them, and the immunoglobulin class of anti take advantage of the fact that the latter have bodies in complexes. No current technique can decreased solubility at 4 ec, at low ionic strength, demonstrate the presence or absence of immune or in salting-out conditions. These approaches complexes. Perhaps some of these techniques, both have, however, been unsatisfactory for several physicochemical and biologic, should be combined reasons, including the fact that under these condi but such a combination would be too cumbersome tions many unwanted proteins, such as antibodies for routine diagnoses. Further research is obviously with rheumatoid factor activity and Clq, tend to needed. precipitate. INVOLVEMENT OF COMPLEMENT BY CLASSICAL OR ALTERNATIVE PATHWAY Biologic Methods If fluorescent antibodies directed against the in Several biologic methods have been used to dividual components of the classical or alternative detect circulating immune complexes. C1q has pathway are used, the binding of these components been used to detect immune complexes because it at the site of the lesions can be followed. This can precipitate with them in agarose gel. Asghar, technique, which does not differ from that used to Faber, and Cormane [15], however, have shown in detect antigen or antibody, can be used with the serum of a patient with allergic vasculitis that FTC-labeled antisera to any of the complement C1q gave a strong precipitation line with some components. Immunofluorescence has been espe unidentified material which could not be shown to cially useful with anti-C3 since C3 is present in the be an immune complex. Similarly, precipitation of immunologic lesions in largest amounts. C4, C5, complexes by isolated monoclonal rheumatoid fac and Clq have also been detected in lesions. In all of tors may give a false positive reaction. the diseases listed in Table III concomitant deposi A comparatively more sensitive and reproduci tion of complement has been observed. Deposition ble procedure for detecting soluble immune com of properdin and C3 proactivator have been ob plexes has been reported [16]. Radiolabeled C1q is served in kidney biopsies of systemic lupus ery reacted with sera containing immune complexes. thematosus (SLE) nephritis, chronic membrano Both free and complex bound (1251) Clq are sepa proliferative glomerulonephritis, and other dis rated by selective precipitation with polyethylene eases [20], but such data with these or other glycol. Complex bound radioactivity is the mea components of alternative pathway are scarce in sure of immune complexes. This method is more skin diseases. sensitive than agarose gel precipitation but may Evidence of in vivo complement activation by have similar drawbacks of reaction with nonim either of the pathways is the detection of fixed mune Clq reactants. In addition, the different Clq components, decreased levels of the components levels in serum samples might have some effect of alternative or classical pathway, and the forma since higher C1q levels may cause more competi tion in vivo of breakdown products of components tion with radioactive C1q than lower levels. More of these pathways. These phenomena have been July 1976 IMMUNODERMATOLOGY 133 studied in detail only with regard to total hemolytic TABLE IV. Some examples of genetic immunologic complement and C3. a 2D, in particular, is found in deficiencies and the defects associated with disease states the circulation of patients with hypocomplemen Disease temic membranoproliferative glomerulonephritis. Deficiency Occasionally in other immune disorders (e.g., Wiskott-Aldrich Syndrome Deficiency in T-cell hypocomplementemic SLE), C3 breakdown has function been observed [21]. Additional research into other Some forms of chronic Deficiency in T-cell skin diseases will probably yield more information. mucocutaneous candidiasis function Total hemolytic activity in the serum can be Some cases of atopy and Defect in or deficiency measured by estimating the degree of hemolysis of a range of disorders of IgA sensitized sheep erythrocytes caused by various Hereditary angioneurotic Defect in or deficiency dilutions of test serum [22]. The individual com edema of Cl-esterase inhibitor ponents can also be estimated by hemolytic meth Arthritis of sex-linked Gammaglobulin deficiency ods which have clearly been described by Lach agammaglo bulinemia mann, Hobard, and Aston [23]. Several hemolytic Lupus-like illness C2 deficiency methods can also estimate the alternative pathway as well as its components. Alternatively, the levels suitable large-scale test for IgE antibody. The of these components or their breakdown products rationale of this technique is that when allergen can be determined antigenically [24,25]. A sample bound to an insoluble matrix such as cellulose is is introduced in the holes at the bottom of the gel incubated with test serum, allergen-polymer-IgE which contains monospecific antiserum. The gel complex is formed. Radioactive anti-IgE can com plate is allowed to stand, as in the Mancini tech bine with this complex, and the radioactivity in nique, or electrophoresed. The area of ring or the the insoluble matrix gives the amount of IgE height of the precipitate is the measure of antigen present in the serum. concentration or, in this case, of the concentration of component of these pathways. DETECTION OF CELLULAR HYPERSENSITIVITY AND STUDIES OF ANCILLARY CELLS IMMUNODEFICIENCIES AND OTHER GENETIC Many authorities still regard skin tests as the ABNORMALITIES only valid measure of cutaneous delayed hypersen The term immunodeficiency encompasses a sitivity. Several studies, however, report that broad spectrum of abnormalities in the host de many assays of cellular hypersensitivity in vitro fense that ranges from mild to severe and is are equally good procedures. These assays are paralleled by increasing susceptibility to different based on evaluating the ability of lymphocytes to pathogens. Hereditary deficiencies in immuno produce mediators after antigen challenge or chal globulins, complement components, inhibitors of lenge with immunologic equivalent. They include complement system and the constituents of PMN the tests of release of (1) lymphocyte proliferation leukocytes, as well as B- and T-cell function, have factor (LPF), (2) migration inhibition factor been reported. These deficiencies can easily be (MIF), (3) macrophage activator factor (MAF) , tested by the techniques already. described. De and (4) mononuclear chemotactic factor ficiencies in B-cell and T-cell -function can be de (MNLCTX). tected by assaying such functions as antibody and Some of these methods have drawbacks. Since MIF production. antigen-induced proliferation of lymphocytes can In those patients who are only functionally occur without delayed hypersensitivity or with deficient in a particular protein, the protein may Arthus reaction, measuring LPF is not necessarily be defective or have impaired biologic activity; for an expression of delayed-type hypersensitivity. As example, the defective C1-esterase inhibitor in far as using the ability of lymphocytes to produce hereditary angioneurotic edema and the defective MIF to test delayed hypersensitivity is concerned, IgA in some cases of atopy. In such cases, the despite various technical modifications, the pro protein is estimated according to its activity as longed multistep procedures are subjeCt to numer well as antigenically. These activities are too ous technical problems; hence there is considerable nUmerous and diverse to allow us to describe how variation within and between assays. In addition, the activities of the components of the defense competing molecular entities in the supernatant system are determined. Some of the immunodefi fractions which enhance macrophage migration, ciencies and genetic defects, together with the plus the fact that B-cell products such as anti diseases with which they are associated, are listed bodies can inhibit macrophage migration, make it i. n Table IV. hard to believe that the MIF assay really correlates Finally, the' techniques described so far do not with delayed hypersensitivity. Similarly, the tech detect another defect, not in the structure, but in nique which involves assaying MNLCTX can be :he quantity of production. This is the higher levels complicated by the products of complement fac )f IgE in atopic patients. Diagnosis in vitro based tors such as C5a. Furthermore, both T-cell and m IgE levels is becoming a routine analytical B-cell mitogens stimulate T -cells to produce Jrocedure in many laboratories. The radio aller MNLCTX. Even nonmitogenic substances which ;osorbent test (RAST) [25] seems to be the most interact with B-cell membranes such as anti- 134 CORMANE AND ASGHAR Vol. 67, No.1 immunoglobulins, C3 sites, and antigen-antibody complexes are all potent activators of B-cell MNLCTX production [26]. Some investigators have advocated alternative mediator assays, namely, MAF [27]. Antigen stimulated lymphocyte culture supernatants con tain a factor which causes macrophages to take up radiolabeled colloidal gold. This assay is quantita tive, rapid, and sensitive, but gold particles must not be allowed to aggregate; otherwise phagocyto sis occurs and gives a nonspecific result. Another assay for macrophage activation which uses the uptake of radiolabeled glucosamine has been de scribed. Direct immunofluorescence studies of biopsies taken from positive patch tests elicited by various antigens in our laboratory revealed lymphocytes with membrane-bound immunoglobulins through out the dermis and epidermis, particularly in and around the blood vessel walls [28]. With the immunofluorescence technique, we found immu noglobulins and complement-bearing leukocytes and lymphocytes in the lesional skin of specimens of patients with various dermatoses [28,29]. In all the cases of allergic contact dermatitis examined, the percentage of IgD-bearing lymphocytes and PMN -leukocytes was increased. This altered ratio of circulating B-cells and T -cells may indicate an FIG. 2. Eluate from the peripheral lymphocytes of a impaired cooperation of at least these cell popula patient with psoriasis vulgaris with FTC-labeled antihu tions. Like lymphocyte populations, the PMN man IgD to show positive immunofluorescence of the nuclei of the basal cell layer of the uninvolved skin of the leukocyte populations consist of cells with and same patient. without immunoglobulins on the cell membrane in the same ratio as in healthy controls. Another finding observed by the immunofluores ogy. Some of the most obvious gaps are listed cence technique was the occurrence of antibodies below. in eluates of lymphocytes and PMN -leukocytes 1. Various approaches must be used to detect obtained from 5 untreated psoriasis patients [30]. the antigen responsible for the disease state and to These antibodies appeared to be directed mainly uncover the etiology of many diseases. against the nuclei of the basal cell layer (Fig. 2); 2. More accurate methods of detecting immune however, antibodies directed against the nuclei of complexes must be found. epidermal and dermal cells have also been seen. 3. More studies are needed on the components Since the eluted antibodies are directed mainly of the alternative pathways of complement activa against nuclear antigens of dividing epidermal tion and their involvement in disease states. Pro cells and not against the nuclei of all cells, it may duction of antibodies against all of them will be that the nuclear antigens are the nonhistone enable us to estimate them quantitatively. proteins which regulate the growth of the dividing 4. Further studies must be done on the anti epidermal cells [31]. bodies coated on ancillary cells. The lymphocyte eluates of 3 patients with dis coid lupus erythematosus and 2 with pemphigoid REFERENCES contained IgG antibody reactive with normal 1. 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