Modulation of Expression of Delayed Hypersensitivity by ...

Vol. 60, No. 6

INFECTION AND IMMUNITY, June 1992, p. 2522-2528

0019-9567/92/062522-07$02.00/0 Copyright © 1992, American Society for Microbiology

Modulation of Expression of Delayed Hypersensitivity by Mycobacterial Antigen 85 Fibronectin-Binding Proteins HENRY P.

GODFREY,'* ZUOHUA FENG,1 SZABOLCS MANDY,1 KATHLEEN MANDY,' KRIS HUYGEN,2 JACQUELINE DE BRUYN,2 CHRISTIANE ABOU-ZEID,3 HARALD G. WIKER,4

SADAMU NAGAI,S AND HIROMICHI TASAKA6 Department of Pathology, New York Medical College, Basic Science Building, Valhalla, New York 105951; Pasteur Institute of Brabant, 1180 Brussels, Belgium2; Department of Medical Microbiology, School of Pathology, University College and Middlesex School of Medicine, London WJP 7PN, United Kingdom3; Institute of Immunology and Rheumatology, University of Oslo, 0172 Oslo 1, Norway4; and Toneyama Institute of Tuberculosis Research, Osaka City University Medical School, Toyonaka 560, Osaka5, and Department of Bacteriology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 7346, Japan Received 21 January 1992/Accepted 2 April 1992

Although demonstration of delayed hypersensitivity to purified protein derivative of tuberculin (PPD) is an important element in the diagnosis of infection with Mycobacterium tuberculosis, many patients with tuberculosis are anergic. Several possible mechanisms for this specific lack of response have been described. We have now uncovered an additional one. T-cell fibronectin (FN), a lymphokine secreted by activated T cells, is closely associated with the initiation of delayed hypersensitivity reactions. Mycobacterial antigen 85 (Ag85) proteins have been shown to bind to plasma FN. The ability of Ag85 to bind to T-cell FN and modulate expression of delayed hypersensitivity was therefore studied. Purified Ag85 proteins from M. tuberculosis, Mycobacterium bovis BCG, or Mycobacterium kansasii bound to T-cell FN, fibroblast FN, and plasma FN in vitro. Purified 65-kDa heat shock protein (hsp65) from M. bovis BCG did not bind to any FN. Ag85, but not hsp65, inhibited the ability of T-cell FN to agglutinate monocytes in vitro in a dose-dependent manner. In vivo, mixtures of PPD or dinitrophenyl-ovalbumin and purified M. tuberculosis or M. bovis BCG Ag85 proteins elicited significantly smaller delayed hypersensitivity inflammatory reactions in sensitized guinea pigs than did PPD or dinitrophenyl-ovalbumin alone. Purified hsp65 did not inhibit expression of delayed hypersensitivity to PPD or dinitrophenyl-ovalbumin. We suggest that Ag85 proteins could inhibit in vivo expression of delayed hypersensitivity during mycobacterial infections because of their interaction with T-cell FN.

Tuberculosis affects more than 20 million people worldwide and results in an estimated 3 million deaths each year (29). It is on the rise in the United States, with 25,901 new cases reported to the Centers for Disease Control in 1990, a 9.4% increase over the data for 1989 (7). Much of the latest increase in tuberculosis has been linked to reactivation of previously acquired mycobacterial disease in patients with human immunodeficiency virus infection and AIDS (4). Delayed hypersensitivity (DH) to purified protein derivative of tuberculin (PPD) is an important element in diagnosis (6). However, this response is not demonstrable in 15 to 25% of patients with clinical tuberculosis even in the absence of human immunodeficiency virus infection (8). The mechanism of tuberculin anergy is unclear. Suppressive mycobacterial products, suppressive serum factors, suppressive monocyte and lymphocyte populations, redistribution of effector cells to tissue sites of infection, and combinations of these factors have all been suggested to be responsible (11). Few studies have tested for direct action of mycobacterial products on lymphokine mediators of DH. Proteins of the mycobacterial antigen 85 (Ag85) complex are major secretory products of actively proliferating Mycobacterium tuberculosis and account for as much as 30% of proteins secreted in culture (2, 9, 34). The Ag85 complex consists of three genetically distinct, antigenically related proteins of known sequence (Ag85A [31 kDa], Ag85B [30 kDa], and Ag85C [31.5 kDa]) (18). Ag85-related proteins *

Corresponding author.

exist in all mycobacteria (36), and contain both speciesspecific and cross-reactive epitopes recognized by polyclonal anti-Ag85 antisera (30). The function of Ag85 in mycobacterial physiology is unknown. Ag85A and Ag85B bind plasma fibronectin (FN) (1), and Ag85 has been proposed to mediate binding of mycobacteria to FN-coated surfaces (27). FNs are a family of high-molecular-weight glycoproteins found in plasma and tissues that are involved in cell motility and adhesion, embryonic development, regulation of cell morphology, phagocytic function, wound healing, and inflammation (22). One of the FNs, T-cell FN, is produced as a lymphokine by mitogen- or antigen-activated human, murine, and guinea pig T cells and appears to play an important role in the initiation of in vivo DH reactions (14). T-cell FN appears to be an unique FN that is biochemically different from other cellular FNs, with a lower Mr and a smaller mRNA than other cellular FNs (lla, 15). It is also markedly more potent in vitro than other FNs: femtomolar concentrations agglutinate monocytes or peritoneal exudate cells by interacting with multiple classes of cell surface integrins and fucose receptors (10, 14). Several lines of evidence point to T-cell FN involvement in initiating DH reactions in vivo. T-cell FN is rapidly produced following T-cell activation (14). There is a close correlation between the ability of cloned murine CD4+ or CD8+ T-cell lines to secrete this lymphokine after antigenic stimulation and their ability to transfer DH reactions in vivo (14). Finally, monoclonal anti-T-cell FN antibodies interfere with the expression of DH in vivo (25b). 2522

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T-CELL FIBRONECTIN AND ANTIGEN 85 COMPLEX

FNs are produced by alternative splicing of a primary transcript from the single complex FN gene in the genome; much of the molecule is constant, regardless of the cell source, and the individual members of this family have numerous epitopes and properties in common (22). Since Ag85 binds plasma FN, it is reasonable to expect that it might also bind T-cell FN. We therefore determined whether T-cell FN could interact with proteins of the Ag85 complex and whether this interaction could modulate expression of tuberculin DH. Our results show that these secreted bacterial products are able to bind a T-cell lymphokine and diminish the expression of DH.

MATERIALS AND METHODS Mycobacterial proteins and antisera. Components of the Ag85 complex were purified from concentrated culture filtrates of M. tuberculosis, Mycobacterium bovis BCG, and Mycobacterium kansasii (1, 9, 26, 30, 34). Purified preparations of Ag85A from M. tuberculosis [Ag85A(T)] and from M. bovis BCG [Ag85A(B)], Ag85B from M. tuberculosis [Ag85B(T)] and from M. kansasii [Ag85B(K)], and Ag85C from M. tuberculosis [Ag85C(T)] each showed a single band on Western blotting (immunoblotting) with specific antisera. A purified preparation of Ag85 complex from M. bovis BCG [Ag85 complex(B)] contained 90% Ag85A, 5% Ag85B, and 5% Ag85C, as judged by Western blotting. None of the purified Ag85 preparations contained measurable p55 FNbinding protein, as measured by dot immunobinding against rabbit anti-p55 (obtained from T. Ratliff, Washington University School of Medicine, St. Louis, Mo.) (1), by Aurodye staining of fractions for total protein or by Western blot reactivity. Purified recombinant M. bovis BCG hsp65 produced in Eschenchia coli (31) was obtained from R. van der Zee (National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands) and reconstituted in phosphate-buffered saline (PBS), pH 7.2. All purified proteins were aliquoted and stored at -70°C. Production and specificity of rabbit anti-Ag85 complex (34), rabbit antiAg85A (9), and anti-Ag85C (26, 35) have been described. A preparation of goat immunoglobulin G anti-M. bovis BCG culture filtrate proteins was obtained from A. Andersen (Statens Seruminstitut, Copenhagen, Denmark). This immunoglobulin G preparation contained high titers of anti-hsp65 antibody, as judged by Western blotting of recombinant hsp65. Other protein reagents. PPD was purchased (RT52; Statens Seruminstitut). Preparation and purification of 2,4-dinitrophenyl (DNP)11-ovalbumin have been previously described (17). Purified plasma FN was purchased (New York Blood Center, New York, N.Y.). Fibroblast FN was a gift from J. Peters. Purification of T-cell FN from concentrated culture supernatants of normal human peripheral blood mononuclear cells activated with streptokinase-streptodornase has been described previously (15). The concentration of immunoreactive FN in all preparations was assessed by dot immunobinding, using plasma FN as the standard and a cross-reactive rabbit anti-human plasma FN for development; the intensity of reactions was evaluated by reflectance densitometry (15). Dot immunobinding assay. The binding of Ag85 to FN was measured by a modified dot blot technique using a filtration manifold (15). Briefly, 1 ng of FN or bovine serum albumin was adsorbed to nitrocellulose filters (BA85; 0.45-p.m-pore size; Schleicher & Schuell, Keene, N.H.) for 2 h at 37°C, and unreacted sites were blocked overnight at 23°C with 20 mM

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Tris (pH 7.5)-500 mM NaCI-0.05% NaN3-1% milk (TBSmilk). The membrane was replaced in the manifold, incubated with 1 ng of Ag85, hsp65, or DNP-ovalbumin for 2 h at 37°C, washed with TBS-0.05% Tween 20, removed from the manifold, incubated with anti-Ag85 or anti-M. bovis BCG culture filtrate proteins for 1 h at 37°C, reblocked with TBS-milk for 30 min at 23°C, and washed with TBS-0.05% Tween 20. Bound antibody was visualized by alkaline phosphatase technology. The intensity of reactions was evaluated by reflectance densitometry. Suitable positive and negative controls were included with each assay. Monocyte agglutination assay. Duplicate or triplicate aliquots of chromatographically purified human T-cell FN (60 pg) were mixed with aliquots of purified Ag85, hsp65, or PBS, incubated at 23°C for 15 min, and serially diluted in modified RPMI 1640 containing 10% fetal calf serum (lowest dilution, 1/100; total volume, 1.8 ml). Human monocytes, 2.5 x 106 to 3 x 106 cells in 0.2 ml, were added to each assay tube as indicator cells. After 4 to 6 h of incubation at 370C, the tubes were gently swirled to dislodge loosely adherent clumps of macrophages and scored (15). Results are reported as geometric mean titers. Animals, sensitization, and skin testing. Male Hartley guinea pigs (Elm Hill Farms, Chelmsford, Mass.), 400 ± 50 g at the time of sensitization, were injected in multiple sites with a total of 1 ml of Freund's complete adjuvant containing 1 mg of 1-thiocyano-2,4-dinitrobenzene (ICN Biomedicals, Costa Mesa, Calif.) and 1 mg of heat-killed mixed strains of human M. tuberculosis (Ministry of Food, Fisheries and Agriculture, Weybridge, United Kingdom) and were boosted by topical application of 1-thiocyano-2,4-dinitrobenzene multiple times (17). This sensitization protocol yields highly sensitized animals with typical DH responses to intracutaneously injected PPD and DNP-ovalbumin as judged by kinetics and histologic appearance (17). Skin tests on guinea pigs were performed by intracutaneous injection of test materials dissolved in 0.1 ml of sterile PBS. Each tested animal received injections of PBS (negative control) and PPD or DNP-ovalbumin (positive control). The injections were coded and randomized and were given without knowledge of the solutions' content. Injection sites were routinely examined for erythema and edema at hourly intervals for the first 4 h after injection for evidence of immediate and Arthus hypersensitivity, and at 16 to 18 h after injection to document resolution or increase in erythema and edema noted at earlier times. The intensity of any observed erythema or edema during the first 4 h was graded qualitatively on a scale of 1+ to 4+. DH was quantitated by measuring with calipers in two perpendicular directions the extent of erythema at the injection site at 24 h (time of maximal reaction size). Reaction size is reported as the mean area of erythema (in square millimeters) obtained by multiplying reaction measurements and as the mean percentage of the positive control PPD reaction. DH reactions greater than 9 mm2 are considered significant. Punch biopsies (4 mm) of skin reactions were taken from ketamine-anesthetized animals, fixed in glutaral(17). dehyde, embedded, and treated with Giemsa stain Coded sections were evaluated independently by two observers (K.M. and H.P.G.) for deep and superficial dermal cellular infiltrates (17). Deep dermal infiltrates were evaluated qualitatively on a scale of 0 to 4 (0, no infiltrate; 1, minimal infiltrate; 2, moderate infiltrate; 3, heavy infiltrate; 4, very heavy infiltrate and microabscesses). The numbers of in 10 oil superficial dermal infiltrating cells wereas counted mean qualitative power fields. Results are presented

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GODFREY ET AL. TABLE 1. Binding of Ag85 complex proteins to T-cell, fibroblast, and plasma FN' Reaction with FNb

Antibody

Anti-Ag85 complex(B)

Antigen

Ag85 complex(B) Ag85A(T) Ag85B(T) Ag85C(T) hsp65

T cell

52 ± 61 ± 53 ± 62 ± 2±

6 6 7 1

lc

Fibro-

iblao-

73 83 67 64

± ± ± ± 10 ±

30 26 1 1

lc

Plasma

59 64 75 58 14

± ± ± ± ±

11 28 1 1

lc

0 ± 0 0 ± 0 0 ± 0 hsp65 2 ± 1 1 ± 1 0 ± 0 DNP-ovalbumin ture filtrate a Purified preparations (1 ng) of Ag85 complex from M. bovis BCG [Ag85 complex(B)], Ag85A, Ag85B, and Ag85C from M. tuberculosis [Ag85A(T), Ag85B(T), and Ag85C(T) respectively], recombinant hsp65 from M. bovis BCG and DNP-ovalbumin incubated with nitrocellulose-adsorbed FN (1 ng), developed using indicated antibodies and alkaline phosphate methodology, and quantitated by densitometry. Reactivity of Ag85 components, hsp65, or DNP-ovalbumin with bovine serum albumin was