Differential Responses of Human Mononuclear Phagocytes to Mycobacterial Lipoarabinomannans: Role of CD14 and the Mannose Receptor

CD14 is a signaling receptor for both gram-negative bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM) that lacks terminal mannosyl units (AraLAM). In contrast, terminally mannosylated LAM (ManLAM) binds the macrophage mannose receptor (MMRc), although the ability of the MMRc to serve as a signaling receptor has not been previously reported. We compared the abilities of AraLAM and ManLAM to induce distinct responses in two monocytic cell populations, freshly isolated human peripheral blood monocytes (PBM) and monocyte-derived macrophages (MDM). The responses examined were chemotaxis and transient changes in free cytosolic calcium ([Ca²⁺]_in). We found that AraLAM but not ManLAM was chemotactic for both PBM and MDM. Migration of these cells in vitro to AraLAM was specifically blocked by an anti-CD14 monoclonal antibody, suggesting that CD14 mediates the chemotactic response to AraLAM. Subsequently, we found that AraLAM induced a transient rise in [Ca²⁺]_in levels within a subpopulation of PBM but not MDM. This response was blocked by anti-CD14 antibodies. In contrast, ManLAM induced a transient rise in [Ca²⁺]_in levels within a subpopulation of MDM but not PBM. This response was blocked by either anti-CD14 or anti-MMRc antibodies. These data suggest that the MMRc can serve as a signaling receptor and that coligation of both CD14 and the MMRc is required to elicit a specific response. Thus, one response to LAM (chemotaxis) can be elicited solely by engaging CD14, whereas a different response (changes in [Ca²⁺]_in levels) depends on both the differentiation state of the cells and concomitant engagement of CD14 and the MMRc.Uptake of Mycobacterium tuberculosis by mononuclear phagocytes is the first step leading to the development of tuberculosis infection. Following ingestion of the bacilli, the innate immune response against tuberculosis is predominantly directed by activated macrophages (reviewed in reference 17). The cell wall glycolipid lipoarabinomannan (LAM) is one of many mycobacterial products that can affect these immune responses. Vesicles containing LAM are released from phagosomes following macrophage ingestion of M. tuberculosis (36, 38), suggesting that transport of mycobacterial products out of infected macrophages is possible. Furthermore, the presence of anti-LAM antibodies in the sera of tuberculosis patients suggests that LAM is released from infected macrophages in vivo (29). LAM is comprised of a mannose-rich core polysaccharide, containing highly branched arabinofuranosyl side chains, linked via a phosphatidylinositol moiety at the reducing terminus to acyl groups consisting of palmitic and tuberculostearic acids. LAM isolated from pathogenic M. tuberculosis and M. bovis BCG is capped with mannose residues at the nonreducing arabinofuranosyl termini (ManLAM), whereas LAM isolated from rapidly growing avirulent mycobacteria lacks mannose caps at the arabinofuranosyl ends (AraLAM [10, 26]). The presence or absence of terminal mannose residues has been shown to affect the biological activity of LAM. For example, tumor necrosis factor (TNF) production can be induced in macrophages by purified LAM, although AraLAM is 100-fold more potent in this respect than ManLAM (11, 13). Similar results have been observed for interleukin-1 (IL-1) (41), IL-6 (13), chemokines (28, 40), and nitric oxide (28) production. In contrast, both AraLAM and ManLAM induce similar amounts of transforming growth factor β (TGF-β) production in human monocytes (13).Two potential LAM receptors have been identified on monocytic cells. Zhang and colleagues first showed that the release of IL-1β and TNF by LAM-stimulated human blood mononuclear cells could be blocked by an anti-CD14 monoclonal antibody (MAb) (40). CD14 is a 55-kDa glycosylphosphatidylinositol-linked protein expressed on the surface of monocytes, macrophages, microglial cells, and polymorphonuclear leukocytes which serves as a receptor for gram-negative bacterial lipopolysaccharide (LPS) (reviewed in reference 42). Evidence that LAM can bind directly to CD14 was provided by the demonstration that AraLAM could compete for the binding of LPS to soluble CD14 in vitro (27). A role for CD14 in the receptor-mediated uptake of nonopsonized M. tuberculosis was suggested by studies which showed that both anti-CD14 MAbs and soluble CD14 could significantly block the uptake of M. tuberculosis by human microglial cells (25). In contrast, ManLAM has been shown to function as the ligand which is most likely to mediate uptake of M. tuberculosis via the macrophage mannose receptor (MMRc) on human blood monocyte-derived macrophages (MDM) (31, 32). The MMRc is a 162-kDa glycoprotein expressed in abundance on MDM and tissue macrophages but not on freshly isolated peripheral blood monocytes (PBM) (reviewed in reference 33). A role for ManLAM in the MMRc-mediated adherence of M. tuberculosis to MDM was suggested by the finding that an anti-LAM MAb blocked the binding of M. tuberculosis to MDM by up to 49% (31). A subsequent study revealed that differences in the ability of LAM from different strains of M. tuberculosis to mediate adherence to macrophages and to serve as ligands for the MMRc are not solely determined by the presence of terminal mannosyl units (32).In this study, we compared the capacity of AraLAM and ManLAM to regulate different monocytic cell functions in vitro. We found that purified AraLAM, but not ManLAM, could induce a chemotactic response in human PBM and MDM. Antibody blocking and inhibitor data suggest that CD14 serves as a signaling receptor for AraLAM. This chemotactic response is distinct from the abilities of ManLAM and AraLAM to differentially induce a transient rise in free cytosolic calcium levels in the two cell populations. The capacity of PBM to generate a calcium response upon exposure to AraLAM appears to involve CD14, whereas the capacity of MDM to generate a calcium response following exposure to ManLAM requires engagement of both CD14 and the MMRc. Lastly, exposure of MDM to either AraLAM or ManLAM resulted in the selective down-regulation of the function of complement receptor CR3, although LAM treatment did not affect the level of surface CR3 expression.