Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells

Abstract

Nonopsonic invasion of mononuclear phagocytes byMycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a ␤ 2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four-to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.on July 6, 2020 by guest http://iai.asm.org/ Downloaded from MATERIALS AND METHODS Bacteria. M. tuberculosis H37Rv (ATCC 27294) and Mycobacterium smegmatis (ATCC 19420) were maintained on Lowenstein-Jensen slopes. Bacterial stocks required for the in vitro infection assays were generated as follows. The confluent growth of M. tuberculosis on a Lowenstein-Jensen slope was dislodged and suspended in 8 ml of Kirchner's medium, agitated to break up the larger clumps, and allowed to settle for 10 min at room temperature. The top 5 ml was used to inoculate 100 ml of Kirchner's medium and grown with shaking (200 rpm) at 37ЊC for 2 weeks. Fifty milliliters of the 2-week-old c...

The impact of impaired macrophage functions in cystic fibrosis disease progression

“…A similar strong reduction in CD11b expression was reported in CF human monocytes and was associated with a reduced capacity of monocytes to phagocytose opsonized P. aeruginosa [61]. Previous studies have also shown the nonopsonic ingestion of microbial pathogens, such as Mycobacterium tuberculosis [62,63] Leishmania [64], and P. aeruginosa [65] through CR3-mediated phagocytosis. Also defects in the chemoattractants mediated activation of integrins have been documented in CF human and murine monocytes [66].…”

Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism

“…This is consistent with previous ¢ndings of opsonized Rhodococcus equi binding to CHO-Mac-1 cells [40] and suggests that the iC3b binding site in the I domain of CR3 is functional. CR3 on CHO-Mac-1 cells can also mediate non-opsonic binding of Mycobacterium tuberculosis [41], apparently to the L-glucan lectin site of CR3 via a cation-independent mechanism [42]. Taken together, these results suggest that CR3 on CHO-Mac-1 cells was abundantly expressed in an active ligand binding conformation.…”

Macrophage Receptors forMycobacterium tuberculosis

“…As if it were not enough for M. tuberculosis to acquire opsonic C3 peptides by at least two distinct mechanisms, M. tuberculosis can bind to CR3 at two distinct sites on the receptor. Opsonized M. tuberculosis binds CR3 at its C3bi binding domain, and nonopsonized M. tuberculosis uses its endogenous capsular polysaccharides to interact with the ␤-glucan binding site near the C terminus of CD11b (9,10). Experiments using human monocytes and murine macrophages had strongly implied that there is more than one mode of interaction between M. tuberculosis and CR3 (31,37), but unambiguous evidence that nonopsonic (i.e., C3bi-independent) interactions occur was obtained in studies in which CR3 was expressed in a nonmacrophage background, so that endogenous synthesis of C3 by macrophages could not interfere.…”

“…Experiments using human monocytes and murine macrophages had strongly implied that there is more than one mode of interaction between M. tuberculosis and CR3 (31,37), but unambiguous evidence that nonopsonic (i.e., C3bi-independent) interactions occur was obtained in studies in which CR3 was expressed in a nonmacrophage background, so that endogenous synthesis of C3 by macrophages could not interfere. Chinese hamster ovary (CHO) cells stably transfected with CD18 and CD11b bind a strain of M. tuberculosis H37Rv ("CC") in a serum-independent manner, and binding of this strain is not enhanced by fresh human or bovine serum (9). A monoclonal antibody that blocks the C3bi binding site in the I domain of CD11b does not block binding of H37Rv-CC to transfected CHO cells, whereas an antibody to an alternative site within the I domain and an antibody to the C-terminal domain do block binding to CR3 expressed on CHO cells.…”