Applications of staphylococcal protein A in clinical immunology

Clinical Immunology Newsletter vo00ctobt,r

21, t981

Copyright ©

1981 b y G . K. Hall & C o .

I S S N 0197-1859

Applications of Staphylococcal Protein A in Clinical Immunology Gabriel Virella, M.D. Alfredo Espinoza, M.D.

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Protein or agglutinogen A (PA) is the major constituent of the cell wall of 99°70 of the strains of Staphylococcus aureus (45). Most of it is covalently linked to the peptidoglycan of the cell wall, but some is released during cell growth. There is wide variation in the ability of different strains of Cowan I to synthesize PA: The Cowan I strain is one of the best producers, while in strain Wood 46 no PA can be detected (23). Structurally, PA is a single, elongated po.lypeptide chain, with a molecular weight of 42,000 daltons and little or no carbohydrate. Its structure seems to be asymmetrical, with a short COOH terminal segment implanted in the cell wall and a longer NH2 segment exposed to the outside and constituted by four repeating subunits (Figure 1) (60). Its extinction coefficient is 1.65, and its isoelectric point 5.1. The biologic properties of PA are multifold, including complement activation, induction of local and systemic anaphylactic reactions, and antiphagocytic effects due to competition with the macrophage receptors for immunoglobulins (45). The attention of immunologists to staphylococcal PA was initially aroused by an apparently high incidence of natural antibodies to S. aureus in normal human serum, which was eventually proven to be "pseudo-immune," due to nonspecific interactions between

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S. aureus and human immunoglobulins (15).

Interaction of Protein A with Human lmmunoglobulins In characterizing the "pseudoimmune" reaction of PA and human immunoglobulins, Forsgren and Sjoquist (15) found that the reaction involved the binding of immunoglobulin G (IgG) to PA through the Fc fragment. Later investigations showed that this is a high affinity reaction, with an affinity constant of l0 s L/mole (34) and that each intact PA molecule has at least two functional binding sites for IgG (15, 32), although structurally four such sites appear to exist per PA molecule (60). The binding of IgG was further characterized by Kronvall and Williams (40) as being restricted to subclasses I, 2, and 4. IgG3, by not binding to PA, can be easily purified from normal IgG preparations (31, 52), and although Skvaril reported that a portion of normal IgG3 was retained to his PA-Sepharose column (61), this was not observed in

our experiments. By eluting PASepharose columns with a pH 5.0 to pH 2.0 gradient, it is possible to obtain fractions enriched for IgG1 and IgG2, the former showing higher affinity for the substrate (13). About 40070 of IgM proteins were reported to bind to PA-Sepharose columns through the Fc fragment (25), but this observation has not been further explored and is not supported by the results of fractionation of normal and pathologic sera in our laboratory (Virella, unpublished observations). IgA2 proteins of both A2m(l) and A2m(2) aUotypes have been found to bind to PA-Sepharose columns (51, 68) (Figure 2) and to PA-containing staphylococci (63). Human colostral IgA, rich in IgA2 has also been chown to be constituted by two frac-

In T h i s I s s u e Applications of Staphylococcal Protein A . . . . . . . . . . . . . . . . . . . 153 Workshops and Meetings . . . . . . . 160

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Fig. 2. Affinity chromatography of four different IgA proteins in SepharoseProtein A. The arrow shows the change o f buffer from O.1 M sodiunl phosphate, p H 7.2, to 1 M acetic acid. Profiles A and C correspond to two lgA1A proteins, showing practically no binditrg. Profile B corresponds to a IgA2 A2m(1) protein, showing remarkable binding. Binding was also observed with an lgA2 A2m(2) protein, as shown in profile D. (Reproduced from reference 69, with perndssion o f the publishers.)

tions, one reactive with PA and the other unreactive, using PASepharose chromatography (26). Other authors using PA-containing S. aureus found evidence of binding of radiolabeled IgAl proteins (7), but the effects of radioiodination in the binding to PA were not considered. Inganas, in a recent study (33), found evidence that some IgA1 proteins could inhibit the reactivity of a PA-reactive, radiolabeled IgE. According to Inganas, this is a different type of interaction, of low affinity, involving the Fab region. However, Brunda et al. (8) claimed that their reactivity was Fcdependent, since no binding was seen with the Fab~ fragment of one of their reactive proteins. It thus ap-

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pears that the binding of IgA1 protein to staphylococcal PA is probably of low affinity and greatly influenced by the experimental conditions. PA as an immunoadsorbent The affinity of PA for the Fc region of IgG under well-controlled conditions has been used, as stated earlier, as the basis for isolation procedures for IgG subclasses (13, 31, 52). A further expansion of this property has been the use of PASepharose affinity chromatography to separate the Fc and Fab fragments of human IgG (23). There are two potential problems with this approach: a) the Fab fraction could be contaminated with the Fc frag-

ment of IgG3; b) the Fc fraction could be contaminated with undigested IgG. Both problems can be minimized (or totally avoided) by prolonged digestion of the original IgG, since the Fc of IgG3 is very labile (70), and if total digestion is achieved the contamination of the Fc fraction is avoided. It has been shown that immunoglobulin aggregates and IgGcontaining immune complexes (ICs) can bind to PA-containing S. attreus with preference over monomeric IgG (44). Whether this preferential binding can or cannot be successfully exploited under chromatographic conditions is not known, but affinity chromatography on PA-Sepharose has been used by our group as the last step for purification of IgGcontaining ICs (9, 37, 38, 69). Similar approaches have been used by Tucker et al. (65) for the isolation of ICs from rats bearing a Gross virus-induced lymphoma and by Papsidero et al. (50) for the characterization of ICs in the pleural effusion of a breast cancer patient. This approach not only has allowed the unquestionable confirmation of the existence of soluble ICs, but has also provided the investigators with a source of native, "natural" ICs that can be studied for their biologic activities. Studies performed in this laboratory have shown that ICs purified from patients with diabetes mellitus are biologically active and can induce platelet aggregation (67), and that soluble IC purified from a patient with SjSgren's syndrome can activate macrophages and induce their release of enzymes and platelet activation factor (Virella and Espinoza, unpublished observations). Although, in principle, PA reacts preferentially with aggregated IgG and IgG-containing IC, the binding of monomeric IgG is too considerable to allow the direct use of PA in IC-screening techniques. However, FarreU et al. (14) used "P-labeled S. attreus Cowan I to detect IgG-containing IC bound to Clq attached to polystyrene tubes.

PA has also been used as an immunoadsorbent in the case of spontaneous carcinoma of the breast, using plasma from a cancer patient and from dogs. The plasma was circulated over PA in continuous flow extracorporeal circulation and reinfused. This resulted in an acute tumoricidal response, both in the patient (4) and in the experimental animals (64). The precise explanation for this increased cytotoxicity is not known; it could be that blocking antibodies or blocking ICs were removed from circulation. An indirect application of the capacity of PA to adsorb immunoglobulins was published by Bauer et al. (5). According to these authors, enzyme-IgG complexes can be specifically adsorbed by immobilized PA-Sepharose that will not affect free enzyme. This can be a useful approach for the fast interpretation of abnormal isozyme patterns that may be due to the formation of enzyme-IgG complexes. The use of PA as the solid substrate for specific antibody, to be used as an immunoadsorbent, was proposed by Gersten and Marchalonis (18). They were able to covalently couple IgG antibodies to Sepharose PA using dimethylsuberinidate as cross-linking agent. This method has the theoretical advantage of binding the antibody by the Fc portion, leaving the Fab regions free to bind antigen. Another way to use the immunoadsorbent properties of PA is to use S. a t t r e u s as a solid phase adsorbent for IgG-antigen complexes. Such an approach was used by Kessler to purify cell membrane antigens (36). The cell surface membrane proteins were first radioiodinated and solubilized; specific antisera were added next; S. a t t r e u s was used to precipitate the ICs; after washing, the antigens and antibodies were dissociated with 4°7o SDS-6M urea and separated, after reduction and alkylation, by SDSPAGE. This approach allowed the isolation and characterization of membrane immunoglobulin,

B2-microglobulin, H-2 alloantigens, etc. S. aureus Bound Antibodies as I m m u n o l o g i c Probes

In 1972 Kronvall described a rapid slide agglutination method for typing pneumococci based on the bind of S. a u r e u s coated with specific antipneumococci antibodies (39); the same group later adapted the same technique to the serologic grouping of streptococci (10). A very similar approach was used by Harmon et al. to detect antineutrophil antibodies (30). After incubation of fixed neutrophils with sera containing antineutrophil antibodies, they would add S. a u r e u s Cowan I to the slide and reveal the bound IgG by observing the clustering of bacteria around neutrophils by direct microscopy. Both whole S. a u r e u s and purified PA can be labeled with fluorescein or with radioactive tracers; in this way the sensitivity of their use or probes is substantially increased. Fluorescent S. a u r e u s Cowan I was shown to bind to human immunoglobulin-positive lymphoblastoid cell lines and to mouse lymphocytes (21). Similar results were obtained with labeled PA, although it became obvious that the reactivity of normal human lymphocytes was extremely weak and difficult to assess (11). Indeed, it is surprising that any reactivity at all can be detected, since membrane immunoglobulin would not be expected to expose much of their Fc region. Some degree of exposure, however, must exist, particularly in Ig-positive lymphoblastoid cell lines. By labeling PA with radioactive iodine, the sensitivity of the method can be increased sufficiently for the interaction between PA and normal human lymphocytes to be easily detected (11). The two types of tracer PA can also be used to detect heterologous IgG bound to lymphocytes; labeled PA can then be used in indirect techniques to study HLA-antigenic sites, B~microglobulin, and lymphocytespecific antigens (11). In general, the

application of fluoresceinated PA as a secondary reagent to reveal IgG antibody molecules bound to lymphoid cells has found more favor than its use as a primary tag for membrane IgG (1). A similar rationale lies behind the use of '2q-labeled PA to detect antiplatelet antibodies (6, 35). However, since platelets have receptors for aggregated IgG and ICs (49), soluble ICs and nonspecific IgG aggregates will interfere with the detection of antiplatelet antibodies. P A as a Solid-Phase Precipitating Agent

PA-containing staphylococci, added in excess to a mixture of radiolabeled antigen, cold antigen, and IgG antibody, will bind (in a matter of seconds) all free and complexed IgG antibody and precipitate it, replacing a second antibody with the advantages of speed and efficiency (34). One possible limitation of this method is the direct binding of radiolabeled antigens to the bacterium. This would be an obvious problem with antigens such as bovine gamma globulins, but it has also been reported for less obvious antigens, such as one animal tumor antigen (7). A second limitation has been found when radioimmunoassay (RIA) procedures are carried out with serum samples at low dilutions, due to the interference of normal human IgG. Under such conditions, nonspecific immunoglobulins block the binding sites of S. a u r e u s , and there is very little recovery of radiolabeled ICs. This problem can be obviated by using S. a u r e u s precoated with a second antibody (i.e., rabbit anti-goat IgG), an approach successfully used to develop an immunoassay for a-fetoprotein (57). In these circumstances, S. a u r e u s does not replace the secondary antibody but is used as a solid phase carrier for the second antibody. PA-carrying S. a u r e u s in its radiolabeled form can also be used as a general tracer for RIA procedure. In one such procedure, im-

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mobilized antigens are incubated merit that will be activated by the bone marrow cells of a patient prowith the corresponding antibodies, antigen-antibody complexes bound ducing the idiotype-carrying and, after washing, '2q-labeled S. to red cells through PA, inducing paraprotein, proving that the maa u r e u s is added to the mixture. red cell lysis and plaque formation. jority of lymphocytes in both blood In this procedure, similar to a This method quickly became and bone marrow were spontaneradioallergosorbent (RAST) test, popular, particularly for the assay ously producing idiotype-specific labeled S. a u r e u s replaces the second of human antibody-producing B IgM. These authors also demonantibody by binding to the imcells (42, 46, 54) (Figure 3), since strated a low but recognizable mobilized antigen-antibody agthe induction of specific antibody response of the malignant clone to gregates in a direct proportion to production to SRBC (as would be purified protein derivative. the amount of specific IgG antibody required for classic plaque-forming The measurement of "sponbound to the antigen (28). In conassays) is particularly difficult when taneous" plaque-forming cells trast to the RAST procedure, this human cells are used in in vitro follows the same principles, except procedure will predominantly systems. However, the plaquefor the fact that the cells are tested measure IgG antibodies. forming assay does not give results soon after collection, without For use as general tracer, parallel to other indices of B-cell acprecultivation in the presence of a ~2q-labcled PA may have advantivity, such as quantitation of immitogen (17). Although it is not tages over radiolabeled S. a u r e u s by munoglobulins released by the quite clear what is measured under allowing a better defined system. stimulated B ceils, (46). In other these conditions, particularly when According to Langone, radiolabeled words, the stage of differentiation peripheral blood lymphocytes are PA will bind efficiently to comof a plaque-forming cell and an endused, it is likely that the ceils replexed goat and sheep immunopoint plasma cell may be totally difsponsible for plaque formation might globulins. With this approach, the ferent, and assays measuring difbe stimulated B cells in the very early radiolabeled PA can be added after ferent parameters are likely to be afstages of their differentiation. a second goat antibody (anti-human fected by variability in the degree of Protein A has also been proposed IgE, anti-methotrexate) instead of a B-cell differentiation achieved in the as a replacement for heterologous radiolabeled rabbit antiserum and stimulated cultures. anti-heavy chain serum for the used in a wide variety of immunoIn principle, and through the use development of "indirect" plaques assays (41). of adequate antisera, the indirect formed by IgG2-producing cells in Finally, the same principle can be plaque technique could be used to the mouse system (66). adapted to fluoroimmunoassays or analyze the response of B cells in Finally, Smith and Hammarstrom enzyme immunoassays by labeling more detail. Hammarstrom et al. demonstrated that PA plaque assays PA with fluorescein isothiocyanate (29) used anti-idiotype antibody ancan be adapted to the detection of or with a suitable enzyme and using tisera to study spontaneous plaquecells secreting any immunogenic it as a replacement for'similarly forming cells from the blood and protein by developing an assay for labeled anti-gamma globulin antisera (2, 3). The use of PA seems to increase the specificity of the proEditors: Herman Friedman, Mario R. Escobar, and Noel R. Rose cedure, although at a cost of detecting only (or predominantly) IgG Editorial Committee: Charles D. Graber, Ph.D., Medical University of South Carolina; John R. antibodies (2). Kateley, Ph.D., Edward W. Sparrow Hospital Association; Bruce S. Rabin, M.D., Ph.D., University

Plaque-Forming Assays Using SRBC Coated with PA In 1976, Gronowicz et al. (24) described the adaptation of the plaque-forming assay to detect immunoglobulin-secreting cells in a murine model. Basically, this is a very simple assay: a suspension of B cells is mixed with molten agar, an anti-immunoglobulin reagent that will form complexes with immunoglobulins secreted by the B cells, PA-coated sheep red blood cells (SRBC) that will bind to the immunoglobulin- anti-immunoglobulin complexes, and comple-

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of Pittsburgh School of Medicine; Robert F. Ritchie, M.D., Foundation for Blood Research, Maine; John L. Sever, M.D., Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health; Steven Specter, Ph.D., University of South Florida College of Medicine; Roy W. Stevens, Ph.D., New York State Health Department Laboratories; Norman Talal, M.D., VA Hospital and University of California Medical Center at San Francisco; Eng M. Tan, M.D., University of Colorado Medical Center; Gabriel Virella, M.D., Ph.D., Medical University of South Carolina.

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lysozyme-secreting leukocytes (62). Rosette-Forming Techniques Using PA-Coated Red Cells SRBC coated with PA can be used to detect mouse or human lymphocytes previously treated with specific IgG antibodies directed to cell membrane antigens (20, 21). Human peripheral blood lymphocytes will also form rosettes with human red blood cells coated with PA. This reactivity can be partially abrogated by the pretreatment of lymphocytes with anti-human F(ab')2, but total abrogation requires treatment with anti-human 7, p, and t~ F(ab'),, indicating that PA is recognizing not only IgG-carrying B cells, but probably some subsets of IgM- and IgD-carrying cells as well (56). The principle of rosette-formation with PA-coated red cells can also be adapted to the separation of lymphocyte subsets. Ghetie et al. (19, 22) adapted their procedure for rosette formation, after exposure of lymphocytes to aggregated IgG or antibodies directed to cell surface antigens, to the purification of the respective cell subsets. After density gradient centrifugation to separate

Fig. 4. Time-course experiments for immunoglobulinsecreting cell (IS(?) numbers and released lg levels. Cells from the same donor were cultivated shnultaneously, stimulated with pokeweed mitogen (A) or S. aureus (B), and harvested on days 3-7for measurements of lSC (plaqueforming cells: PFC, pfc) and extracellular immunoglobulin (lg) levels. (Reproduced from reference 46, with permission o f the publishers.)

the rosetted ceils, the erythrocytes were lysed by osmotic shock or complement addition, and the immunoglobulin-PA complexes were shed off during subsequent cultivation. S. aureus as a B-Cell Mitogen The addition of S. aureus Cowan I or of Sepharose 4B particles coated with PA, but not of soluble PA, to cultures of unfractionated lymphocytes was observed to result in a mitogenic response (16). Enriched B-cell populations showed identical responsiveness to T and B cell mixtures, while enriched T-cell preparations did not respond, suggesting that PA was primarily a B-cell mitogen with very little dependence on T cells for its action. The ability of S. aureus Cowan I to stimulate B cells was confirmed in studies in which the number of antibody-producing cells was determined and the concentrations of released immunoglobulins were measured after variable periods of incubation (46, 53,.63). It was found that the time-response curves were similar with S. aureus and pokeweed mitogen, but S. aureus tended to induce the production of

higher levels of IgM (46, 53) (Figure 4), as perhaps would be expected from a T-independent mitogen. The T dependency of the stimulating effect of S. aureus has been confirmed in several studies in which enriched B-cell and T-cell populations have been stimulated with different mitogens, although it can also be shown that the presence of T cells may potentiate the B-cell response (12, 16, 53, 54, 58), an effect that could be attributed to the leakage of soluble PA from the S. aureus population (58). On the other hand, soluble PA seems to stimulate T cells (27) and induce B-cell proliferation indirectly, through T-cell help (12, 58). Actually, it appears that varying the concentration of PA can stimulate either helper or suppressor T Cells, so that the use of high PA concentrations can induce suppression of the immune response (43). The helper T-cell activity does not require cell division, i.e., it can be observed even when T cells are irradiated or treated with mitomycin (12, 43), but the helper T-cell activity is greater when the T cells are able to proliferate (43). On the other hand, the response of T cells to soluble PA is enhanced by the

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presence o f non-B cells, although a helper effect by the B cells involved in the response to S. aureus Cowan I appears to have been ruled out

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(55). An interesting application of the mitogenic properties o f P A has been its use to promote h u m a n B lymphocyte colony formation (48, 59). Colony formation can also be formed with phytohemagglutinin ( P H A ) and requires the presence o f B cells and irradiated T cells (59). The characteristics o f the B cells induced by either mitogen were found to be markedly different. In P H A induced colonies, the cells had surface IgM a n d / o r IgD, but no cytoplasmic immunoglobulin; while in PA-induced colonies, the cells had cytoplasmic immunoglobulin and showed a sequence of IgM-IgG and IgA production (48). These results could correspond to the stimulation of different B-cell subsets or to a more intense drive towards differentiation in the presence of PA. Conclusion In little more than a decade, staphylococcal protein A has emerged as a m a j o r immunologic reagent, with a multitude of applications. Some have taught us about immunoglobulin structure, others about lymphocyte diversity, and others have represented significant improvements in research and diagnostic procedures. All in all, the story o f PA illustrates an exemplary evolution from serendipitous observations to m a j o r applications. We hope that curiosity about why and how P A works will not be extinguished and that the future will bring us even more interesting findings and applications. of this work were supported by the State of South Carolina Appropriations for Medical Research and by The Kroc Foundation.

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References 1. Ades, E. W., et al. 1978. Antiserums for immunofluorescent enumeration of human T lymphocytes utilizing fluoresceinated

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Workshops and Meetings The Second Annual International Congress for Interferon Research. San Francisco, California. October 21-23, 1981. Contact: William E. Stewart II o f the Sloan Kettering Institute for Cancer Research. Hyatt on Union Square, 345 Stockton Street, San Francisco, CA 94108. Tel. (415) 398-1234. Rapid Methods in Diagnostic Microbiology and Immunology. Causeway Inn, Tampa, Florida. October25-27, 1981. Contact: Dr. Steven Specter, Department of Medical Microbiology and Immunology, College o f Medicine, University o f South Florida Medical

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