Faber, J. E. R. Thole, and M. Harboe. 1992.Heterogeneity of monoclonal antibody-reactive epitopes on mycobacterial 30- kilodalton-region proteinsand the ...
INFECrION AND IMMUNITY, May 1993, P. 1835-1845
Vol. 61, No. 5
0019-9567/93/051835-11$02.00/0 Copyright © 1993, American Society for Microbiology
The Mycobacterial Secreted Antigen 85 Complex Possesses Epitopes That Are Differentially Expressed in Human Leprosy Lesions and Mycobacterium leprae-Infected Armadillo Tissues ANURA RAMBUKKANA,' JAN D. BURGGRAAF,1 WILLIAM R. FABER,' MORTEN HARBOE,2 PETER TEELING,' SUZE KRIEG,' AND PRANAB K. DAS1* Departments of Dermatology and Pathology, Academic Medical Center, University ofAmsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands, 1 and Institute of Immunology and Rheumatology, University of Oslo, N-0172 Oslo 1, Norway2 Received 22 October 1992/Accepted 21 December 1992
The granulomatous skin lesions in leprosy are thought to be initiated by the immune response to certain antigens of the causative agent, Mycobacterium leprae. The antigen 85 complex is one of the major targets in the immune response to M. leprae infection. In the present study, a panel of previously characterized monoclonal antibodies (MAbs) (3A8, Rb2, A4g4, A2hll, Pel2, and A3c12) reacting with different epitopes of the 85 complex proteins of Mycobacterium tuberculosis and M. leprae was employed in a comparative immunohistological analysis to demonstrate the in situ expression of 85 complex antigenic epitopes in leprosy lesions across the clinical spectrum and in M. leprae-infected armadillo liver tissues. These MAbs showed a heterogeneous staining pattern in a given leprosy lesion. In highly bacilliferous borderline and lepromatous leprosy lesions, MAbs Rb2, A4g4, A2hll, and Pel2 stained clear rod-shaped M. leprae bacilli within macrophages, and the degree of staining correlated with the bacillary index of the lesion. On the other hand, MAbs 3A8 and A3c12 staining was mostly seen as a diffuse staining pattern within interstatial spaces and on the membranes of the infiltrated cells but not the bacilli. In paucibacillary borderline and tuberculoid leprosy lesions, only 3A8, Rb2, and A3c12 showed distinct staining in association with infiltrates in the granuloma. None of these MAbs showed any detectable reaction with control nonleprosy skin lesions, while MAb A3c12 positively stained the granulomas of both leprosy and control specimens. In situ reactivity of these MAbs with M. keprae-infected armadillo liver tissues also showed a heterogeneous staining pattern. Interestingly, a clear difference in expression of these epitopes was observed between armadillo tissues and human leprosy lesions. By immunogold ultracytochemistry, we further showed the differential localization of these MAb-reactive epitopes on the cell surface, in the cytosol, and at the vicinity of M. leprae within Kufper cells of armadillo liver tissues. Our results indicate that these antigenic epitopes of the antigen 85 complex are differentially expressed in leprosy lesions and infected armadillo tissues and that they could be target determinants in the immunopathological responses during M. leprae infection.
The focal point of the immune responses to Mycobacterium leprae is the formation of granulomatous skin lesions (8, 14, 21, 22). Most of the tissue damage seen in leprosy is thought to be due to the patient's own immune response to the antigens of M. leprae bacilli (9-11, 22, 26). It is believed that mycobacterial antigens are continuously being released into the tissues and could be responsible for the formation and persistence of the granulomatous lesions (9-11). However, which M. leprae antigens are involved in the granuloma formation or in the immunopathology of leprosy lesions remains unknown. Proteins of the antigen 85 complex, which are referred to as 30-kDa region proteins, are known to be the major secreted proteins of mycobacteria. They are considered to be the important target antigens in both B-cell and T-cell responses during infections with M. leprae and Mycobacteium tuberculosis (2-5, 13, 16, 25, 27, 30, 32, 33). Our recent study has suggested that the antigen 85 complex may also play a significant role in the immunopathology of leprosy (20). By using a novel monoclonal antibody (MAb) 3A8 specific for 85 complex proteins in the immunohistochemis-
try of leprosy lesions, we have shown that this MAb-defined epitope is expressed in leprosy lesions in a pattern that is variable across the clinical spectrum of leprosy (20). It is known that the immune response to complex microbe-like M. leprae involves multiple epitopes. Therefore, it is essential to study the involvement of different epitopes of the antigen 85 complex in leprosy lesions in order to gain further insight into the role of the 85 complex proteins in the pathology of leprosy. More recently, we have characterized further a panel of new MAbs directed to different epitopes of the 85 complex proteins ofM. tuberculosis and M. leprae (18). In the present study, using these MAbs, we have carried out a detailed comparative immunohistochemical analysis of skin lesions of patients with various forms of leprosy. Moreover, since the armadillo is considered to be an experimental animal model for human leprosy and the 85 complex is known to be a major immunogen in vivo during M. leprae infection in armadillos (33), we have also investigated in parallel the in situ expression of these epitopes of the 85 complex in M. leprae-infected armadillo liver tissues. Here, we demonstrate that different epitopes of the antigen 85 complex are differentially expressed within the lesions of lepromatous and tuberculoid leprosy lesions as well as in M. lepraeinfected armadillo tissues.
* Corresponding author. 1835
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TABLE 1. In situ reactivity of MAbs directed to different epitopes of the antigen 85 complex as analyzed by immunohistochemical staining No. of positive biopsies/no. of biopsies tested (intensity of staining) with MAba:
Tissue
Leprosy lesion (+++) TI (untreated; BI = 0) 3/3b BT (untreated; BI = 0 to 1+) 515b (++ to BL/LL (untreated; BI = 4+ to 6+) 6/6e(++ to RR (treated; BI = 0) 3/3b (++ to ENL (treated; BI = 1+ to 4+) 2/2b (++ to
A4g4
Rb2
3A8
A2hll
Pel2
A3c12
2/3C (++) 0/3 +++) 2/5c (+/- to +) 0/5 +++) 5/6f(+++) 2/6f(+++) +++) 1/3c (+/-) 0/3
3/3d (+ to ++) 0/3 0/2 4/4" (+ to ++) 0/4 0/3 5/6f(+++) 4/5f(+++) 4/4d (++ to +++) 2/2d (+ to ++) 0/3 0/2
+++) l/2C (+1-)
0/2
0/2
0/1
1/1d (+ to ++)
0/5
0/3
0/3
0/2 0/2
0/2 0/3
0/2 0/2 0/1
4/4d (+ to + +) 2/2d (+ to ++)
1/1l (++++) 1/ll (+ + +) l/lf (+ + +) 111i (+ +)
Other skin lesion Sarcoidosis Borreliasis (Lyme disease) Psoriasis
0/5 0/3 0/5
0/3 0/5
M. leprae-infected armadillo liver
1/1g (+ + +)
1lif (+ + +)
4/4d (+ to ++)
a Results are averages of the individual positive staining reactions of the specimens. The positive staining was graded as weak (+/-) to maximum brightness
(+b+ + +); 0 indicated no staining.
Positive staining was mostly associated with infiltrating cells in the dermal lesions, whereas in patients with reversal reaction MAb 3A8 showed staining on epidermal cells in addition to the dermal infiltrating cells. c Positive staining was seen as a diffuse pattern within the granuloma. d MAb A3c12 stained the cellular infiltrate in the dermis and the basement membrane of the epidermis in all leprosy and nonleprosy skin lesions as well as in normal skin specimens tested. e Diffuse staining with granular appearance and occasional membrane and cytoplasmic staining in the dermal infiltrates. f Only the clear rod-shaped bacilli were stained. All positive MAbs showed almost similar intensities of staining. However, MAb A4g4 detected significantly fewer bacterial cells only in human lesions than did the other 3 MAbs. g Reaction was mostly seen as extracellular staining. h Bright fragmented and rod-shaped pattern of staining, which is quite different from the staining pattern seen in lepromatous leprosy lesions, was seen. i Reaction was mostly seen as cell membrane staining.
MATERLALS AND METHODS Patients and tissue specimens. The lesional skin biopsies from patients (n = 19) with various forms of leprosy in this study were similar to those used in our previous study (20). All the patients were classified by using the clinicopathological criteria of Ridley and Jopling (24). Table 1 summarizes the details of the leprosy lesions studied. Liver specimens from an M. leprae-infected armadillo were obtained from T. A. Eggelte, Royal Tropical Institute, Amsterdam, The Netherlands. All tissue specimens were stained with the conventional Fite-Faraco-Wade stain for the determination of the bacillary index (BI), which is a logarithmic scale of the number of mycobacteria present in sections of skin biopsies (23). As control specimens, skin lesion specimens from patients with sarcoidosis (n = 5), psoriasis (n = 5), or cutaneous borreliasis (Lyme disease) (n = 3) as well as normal skin biopsies (n = 6) were included. Preparation, purification, and labeling of MAbs. Preparation and characterization of MAbs reacting with the 30-kDa region proteins of the antigen 85 complex have been described in detail in our previous reports (18, 19). Briefly, spleen cells from BALB/c mice immunized with gel-purified 30-kDa region antigen fractions from an M. tuberculosis sonicate were fused with the Ag8-653 BALB/c myeloma cell line. Antibody activity in hybridoma supernatants was determined by using the same 30-kDa region antigen fraction. Selected hybridomas were cloned by limiting dilution, and stabe clones (MAbs) were further characterized by dot blot enzyme-linked immunosorbent assay (ELISA) with purified proteins of the antigen 85 complex as well as recombinant fusion proteins of the M. leprae 30/31-kDa 85 complex (18). Of 13 anti-30-kDa MAbs, six MAbs, 3A8 (immunoglobulin Gl [IgGl]), A4g4 (IgGl), Rb2 (IgGl), A3c12 (IgG2a), Pel2 (IgGl), and A2hll (IgGl), directed to six different epitopes of the antigen 85 complex of M. tuberculosis and M. leprae
were selected in this study after preliminary immunohisto-
chemical analysis of leprosy lesion specimens and M. lepraeinfected armadillo tissues. In most cases, culture supernatants containing MAbs were grown in bulk and concentrated at least up to 10 times with the Minitan ultrafiltration system (Millipore, Bedford, Mass.), whereas ascitic fluids were made from some clones. Immunoglobulins were partially purified from most of these preparations by ammonium sulfate precipitation, while some of the MAbs were affinity purified by chromatography on PERFEX 35S recombinant protein A columns (Du Pont, Wilmington, Del.). Affinity-purified MAbs were conjugated with fluorescein isothiocyanate (FITC) according to our previous report (20). Purification of the proteins of the antigen 85 complex. The individual proteins of the secreted antigen 85 complex, i.e., 85A (MPT44), 85B (MPT59), and 85C (MPT45), were purified from concentrated M. tuberculosis H37Rv culture fluid by a combination of ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on DEAESepharose as described in detail previously (13). Immunolabeling of tissues. (i) Immunoenzyme single and double staining. Tissue specimens were snap frozen in liquid nitrogen and stored at -70°C. Frozen sections (6 ,um thick) were cut, dried, fixed in cold acetone, and incubated with a preevaluated optimum dilution of anti-antigen 85 complex MAbs. MAbs 3A8 and Rb2 were used either as ascitic fluids (dilution 1:100) or as affinity-purified antibodies (dilution 1:50); MAbs A4g4, A2hll, and Rb2 were used as partially purified immunoglobulins, whereas MAb A3c12 was employed as a concentrated culture supematant. Each of the last four MAbs was diluted at 1:10. Negative control sections were incubated in phosphate-buffered saline (PBS; pH 7.4). A two-step peroxidase-conjugated rabbit anti-mouse antibody-staining system with 3-amino-9-ethylcarbazole as the
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FIG. 2. Representative examples showing differential in situ expression of epitopes of the antigen 85 complex in lepromatous and tuberculoid leprosy lesions as analyzed by immunohistochemistry with MAbs directed to different epitopes of the antigen 85 complex. Immunoperoxidase staining of serial sections of an untreated bacilliferous LL lesion (BI = 6+) (A) and an untreated paucibacillary TT lesion (BI = 0) (B) with MAbs 3A8 (Al and Bi), Rb2 (A2 and B2), A4g4 (A3 and B3), A2hll (A4 and B4), and A3c12 (A5 and B5). MAbs Pel2 and A2hll showed identical staining. Note the MAb A3c12 staining with the basement membrane as indicated by arrows (also note the strong melanin-containing basal layer of the epidermis in the skin lesion of panel B, and this is not due to any staining with MAbs). All sections were counterstained with hematoxylin. Original magnifications, x560 (A) and x300 (B).
in order to clarify the antigen 85 complex-specific immuno-
staining, an inhibition experiment was performed by preincubation of MAbs with either purified antigen 85 complex or total culture fluid of M. tuberculosis (all MAbs included in this study reacted only with 30/31-kDa doublet protein in the culture fluid, which corresponds to the 85 complex). Staining of either rod-shaped bacilli or the granuloma is greatly inhibited after absorption of the MAbs with antigen 85 complex or 30-kDa region protein, indicating that the latter is responsible for immunostaining in leprosy lesions. In view of the specific staining of MAb 3A8 on cells infiltrating the granulomas of BTITT patients, we performed immunoenzyme double staining to ascertain the 3A8+ cell types in the granuloma. For this purpose, FITC-labeled
affinity-purified MAb 3A8 and unlabeled anti-CD68 MAb (macrophage marker) were used. Figure 3 illustrates a representative example showing the simultaneous localization of many 3A8-anti-CD68 double-positive cells in the granuloma of a BT patient (BI = 0), indicating that the 3A8 epitope of the antigen 85 complex is expressed on macrophages in the lesions. It should also be noted that we have previously shown that these 3A8-anti-CD68 double-positive cells are in close contact with infiltrating T cells positive for CD3+ and T-cell receptor ca3 cells (20). For MAb Rb2, we have performed single staining on serial sections of Rb2-positive BT/TT lesions with MAbs to macrophages and T cells. These results indicate that most areas in the infiltrates stained for Rb2 were also positive for CD68+ macrophages
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representative double immunoenzyme staining of a granulomatous BT lesion with FITC-labeled MAb 3A8 (red; antigen 85 [anti-CD68] or a distinct purple [3A8+ CD68+ cells]) in the granuloma. Some of the double-positive cells are indicated by the arrows. Original FIG. 3. A
complex) and unlabeled anti-CD68 MAb (blue; macrophage marker). Note the many double-positive cells (both red [MAb 3A8] and blue
magnification, x650.
and CD3+ T cells as well as T-cell receptor ac43 cells (data not shown). Moreover, many cells in the granuloma were found to be HLA-DR positive (data not shown). In situ expression of the 85 complex antigenic epitopes on M. leprae-infected armadillo tissues. (i) Immunoperoxidase staining. Figure 4 illustrates the variable in situ expression
patterns of four different MAb-reactive epitopes of the 85 complex with serial sections of liver tissue of an armadillo infected with M. leprae. MAbs Rb2, A2hll, Pel2, and A4g4 showed heavy bacillary staining in the armadillo tissues. However, M. leprae staining by MAb A4g4 appeared to be somewhat different from that by the other three MAbs, as the latter strongly stained clear rod-shaped M. leprae bacilli while most of the A4g4 showed a bright fragmented form of bacillary staining (Fig. 4C). An interesting observation is that MAb A4g4 stained numerous bacilli in the armadillo tissues compared with highly bacilliferous human BL/LL lesions (Fig. 2A3 and 3C). On the other hand, MAb 3A8 compared with other MAbs showed a clear extracellular staining pattern throughout the infected armadillo tissue (Fig. 4A). MAb 3A8 staining was not found in the form of bacilli or the fragmented form although the liver tissue was heavily infected with M. leprae. The staining patterns by MAbs A4g4 and 3A8 showed clear differences in epitope expression in human BL/LL lesion specimens and infected
armadillo tissues. In contrast, MAb A3c12 showed membrane staining on some cells, most probably the Kupffer cells. This MAb did not stain any bacteria. (ii) Immunogold ultracytochemistry. In view of the differential bacterial staining patterns by most MAbs, we performed the ultrastructural in situ localization of these epitopes within the M. leprae bacilli in armadillo tissues. Figure 5A shows a Kupffer cell of armadillo liver infected with numerous M. leprae bacilli (a negative control). In situ gold labeling of serial sections of the same tissue with MAbs A4g4 and Rb2 is shown in Fig. 5B and C. The site of gold labeling by MAb A4g4 could be seen mostly in the vicinity of M. leprae bacilli or within the cytoplasm of the Kupffer cell and occasionally on the cell surface of the bacilli (Fig. 5B and C). No gold particles were found inside the bacterial cell. In contrast, a significant number of gold particles could be clearly detected by MAb Rb2 on the cell wall surfaces of M. leprae bacilli residing within the armadillo Kupffer cell (Fig. 5D, E, and F). Compared with A4g4, MAb Rb2 showed a significant number of gold-labeled particles within the bacilli (Fig. 5F). Immunogold labeling by these MAbs was evaluated as specific binding since no gold particles were detected in the control experiments (Fig. 5A). For both MAbs 3A8 and A3c12, we were unable to locate gold labeling in either armadillo tissues and leprosy lesion speci-
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