Differential responses of human mononuclear phagocytes to mycobacterial lipoarabinomannans: role of CD14 and the mannose receptor

Abstract

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.

FIG. 1

PBM and MDM chemotactic responses to AraLAM and ManLAM. Cell migration was measured in modified Boyden chambers, and data are expressed as a percent of unstimulated (control) migration in medium alone. (A) Forty-eight-hour MDM migration induced by fMLP (100 nM), AraLAM (1 μg/ml), AraLAM plus anti-LAM MAb CS-35 (1 μg/ml), and ManLAM (1 μg/ml). The anti-LAM MAb alone had no effect on cell migration (not shown; n = 4 separate experiments from different donors). (B) Fresh (0-h) PBM migration induced by fMLP (100 nM), AraLAM (1 μg/ml), and ManLAM (1 μg/ml). Asterisks denote statistically significant migration compared with controls (P < 0.05, n = 3).

FIG. 2

Blocking of MDM and PBM chemotactic responses to AraLAM by anti-CD14 and RSLA. MDM (A) and PBM (B) migration to AraLAM (1 μg/ml) was measured in Boyden chambers as described for Fig. 1. The abilities of the anti-CD14 MAb 3C10 (1 μg/ml) and the LPS antagonist RSLA (1 μg/ml) to inhibit this response were determined. Cells were preincubated with the anti-CD14 MAb or RSLA for 5 min at 4°C prior to LAM treatment. Anti-CD14 or RSLA alone had no effect on macrophage migration (not shown). Asterisks denote statistically significant migration compared with controls (P < 0.05, n = 3).

FIG. 3

[Ca²⁺]_in response to AraLAM or ManLAM in 0- and 48-h monocytes/macrophages. Cells were loaded with the fluorescent probe Indo-1 and then stimulated with either fMLP (100 nM; open circles), AraLAM (1 μg/ml; squares), AraLAM plus anti-CD14 (each at 1 μg/ml; closed circles), or ManLAM (1 μg/ml; diamonds). Changes in [Ca²⁺]_in were continuously recorded as changes in Indo-1 F405/485 ratio (proportional to [Ca²⁺]_in by FACS. Representative tracings from each series of experiments are illustrated. (A) Fresh (0-h) PBM demonstrated brisk responses to fMLP stimulation and to AraLAM; there was no response to ManLAM (n = 5). Gating of responsive cell subpopulations revealed that 13.6% of PBM responded to AraLAM (not shown). (B) With 48-h MDM, [Ca²⁺]_in responses to fMLP were similar to those of PBM. ManLAM induced a slow [Ca²⁺]_in signal, whereas there was no response to AraLAM (n = 5). Gating of responsive cell subpopulations revealed that 12.4% of MDM responded to ManLAM (not shown).

FIG. 4

Inhibition of the MDM [Ca²⁺]_in response to ManLAM by an anti-MMRc MAb. Indo-1-loaded MDM were stimulated with fMLP (circles) or ManLAM (open diamonds), and changes in [Ca²⁺]_in (F405/485 ratio) were continuously recorded by FACS as described for Fig. 3. In addition, aliquots of cells were reacted with an anti-MMRc MAb (1 μg/ml) for 5 min on ice prior to warming (37°C) and ManLAM stimulation (closed diamonds). Data illustrated are representative of three separate experiments from different donors. Addition of the anti-MMRc MAb alone to MDM at 37°C had no effect on resting cells or on subsequent fMLP-induced changes in [Ca²⁺]_in (not shown).

FIG. 5

Surface phenotypes of PBM and MDM incubated without and with LAM. (A) Surface phenotype of macrophages incubated in medium alone. Fresh PBM (0 h; open bars) and 48-h MDM (hatched bars) on ice were stained for CD14, CD11b, and MMRc and analyzed by FACS. The percentage of labeled cells is shown on the ordinate. The asterisk denotes statistically significant changes in receptor expression (P < 0.05, n = 4). (B) Effect of LAM on CD14 subpopulation phenotype. MDM incubated for 48 h (37°C, 5% CO₂) in medium alone, AraLAM (1 μg/ml), or ManLAM (1 μg/ml) were stained with FITC-labeled anti-CD14 MAb TUK4 (0°C) and analyzed by FACS. Representative histograms obtained in assays using untreated (medium alone; top histogram) and AraLAM-treated (bottom histogram) MDM stained with the anti-CD14 MAb and with an isotype control MAb are illustrated. ManLAM incubation resulted in a CD14 staining pattern similar to that obtained after incubation with AraLAM (histogram not shown). (C) Percentage of cells expressing high levels of surface CD14 (CD14^hi) following 48 h of treatment with AraLAM or ManLAM compared with cells incubated in medium (n = 4).

FIG. 6

Effect of LAM incubation on MDM [Ca²⁺]_in response to fMLP. MDM were incubated for 48 h (37°C, 5% CO₂) without (circles) or with AraLAM (1 μg/ml; squares) or ManLAM (1 μg/ml; diamonds) prior to determination of the intracellular [Ca²⁺] response to fMLP (100 nM). Changes in [Ca²⁺]_in are shown as in Fig. 3 and 4. Data illustrated are representative of four separate experiments.

FIG. 7

Effect of LAM incubation on complement receptor CR3 function of MDM. MDM were incubated without (control) or with AraLAM or ManLAM (each at 1 μg/ml) for 48 h (37°C, 5% CO₂) and were then assayed for binding of opsonized SRBC by light microscopy as described in Materials and Methods. Asterisks denote a statistically significant reduction in binding compared with control cells (P < 0.05, n = 4).