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. 1997 Jun 2;185(11):1987-95.
doi: 10.1084/jem.185.11.1987.

Regulation of interleukin-12 by complement receptor 3 signaling

T Marth  1 , B L Kelsall
Affiliations

Regulation of interleukin-12 by complement receptor 3 signaling

T Marth et al. J Exp Med. .

Abstract

Complement receptor type 3 (CR3, CD11b/CD18) serves as a receptor for a number of endogenous ligands and infectious organisms, and is involved in adhesion and host defense functions. Here, we report that signaling via CR3 plays an important role in regulating production of interleukin-12 (IL-12), a key mediator of cell-mediated immunity (CMI). We demonstrate with a variety of stimuli a dose-dependent, specific downregulation of IL-12 secretion by human monocytes in vitro after exposure to antibodies to CR3 (anti-CD11b and anti-CD18), as well as to the natural CR3 ligands, iC3b, and Histoplasma capsulatum. CR3 antibodies also suppressed interferon-gamma (IFN-gamma) production in cultures of human peripheral blood mononuclear cells (PBMC). We determined that one mechanism by which CR3 antibodies may suppress IL-12 production is by the inhibition of IFN-gamma-induced tyrosine phosphorylation. Finally, in a murine model of IL-12-dependent septic shock, we provide evidence that administration of CR3 antibodies leads to suppression of IL-12 and IFN-gamma in vivo. Our studies thus define a novel role for CR3 in regulating CMI functions via IL-12.

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Figures

Figure 1
Figure 1
Effects of various integrin antibodies on secretion of IL-12 and other cytokines by human monocytes. (A–C) Production of (A) heterodimeric IL-12 (p70) and (B) monomeric IL-12 (p40) in cultures of highly purified human monocytes as determined by ELISA is suppressed by antibodies to CD11b and CD18 in a dose dependent manner, but not by anti-CD11a and modestly by anti-CD11c, when stimulated with SAC (0.01%) and IFN-γ (1 μg/ml). Secretion of TNF-α (C) in the same culture supernatants is not altered by such antibodies. Data in A–C represent means of duplicates from one experiment and are representative of five experiments. (D and E) Suppression of IL-12 p70 by anti-CD11b (clone LM2/1) in a dose-dependent fashion after stimulation with LPS (1 μg/ml) plus IFN-γ (1 μg/ml) and CD40L trimer (3 μg/ml) plus IFN-γ (1 μg/ml). One of three experiments is shown.
Figure 2
Figure 2
Monocytic IL-12 production induced by various stimuli is suppressed by antibodies to CD11b. Secretion of IL-12 as determined by ELISA in cultures of human monocytes after stimulation with (A) LPS and IFN-γ, (B) SAC and IFN-γ, and (C) LPS alone (1 μg/ml), SAC alone (0.01%), SAC plus anti–IFN-γ (10 μg/ml), SAC plus IFN-α (10 ng/ml), and CD40L trimer (3 μg/ml) plus IFN-γ (1 μg/ml). Similar results were obtained with LPS plus increasing doses of IFN-γ, SAC plus increasing doses of IFN-γ, SAC plus TNF-α, TNF-α, and SAC plus anti–IL-10 (data not shown). One of three experiments is shown.
Figure 3
Figure 3
Inhibition of monocyte IL-12 production by a natural ligand to CR3, iC3b-coated SRBC. iC3b-SRBC (2 × 107/ml) were incubated with human monocytes (2 × 106/ml) 2 h before stimulation with LPS (1 μg/ml) plus IFN-γ (1 μg/ml), SAC (0.01%) plus IFN-γ (1 μg/ml), or CD40L trimer (3 μg/ml) plus IFN-γ (1 μg/ml). IL-10 and TNF-α levels in the same culture supernatants were not significantly altered (data not shown). Data are representative of five experiments.
Figure 4
Figure 4
Secretion of IL-12 p70 by monocytes is suppressed by incubation with heat-killed Histoplasma capsulatum (HC). Heat-killed HC at the indicated concentrations was incubated with human monocytes 2 h before stimulation with LPS (1 μg/ml) plus IFN-γ (1 μg/ml), SAC (0.01%) plus IFN-γ (1 μg/ ml), or CD40L trimer (3 μg/ml) plus IFN-γ (1 μg/ml) (A). (B) IL-10 and TNF-α levels from the same culture supernatants. Data are representative of five experiments, and percent suppression by HC was similar in all instances.
Figure 5
Figure 5
Monocyte IL-12 production is suppressed by antibodies to CD11b (clone LM2/1). Antibodies were bound to culture plates, polystyrene beads, or L cells as described in Materials and Methods. The secretion of IL-12 was determined by ELISA in cultures of human monocytes after stimulation with SAC (0.01%) and IFN-γ (1 μg/ml). One of three experiments is shown.
Figure 6
Figure 6
Prevention of IFN-γ–induced phosphorylation of STAT1 by anti-CR3. Human monocytes (2.5 × 107 in 5 ml per condition) were either untreated (None), treated with anti-CR3 (LM2/1), treated with recombinant IFN-γ (IFN-γ), or treated with LM2/1 followed by IFN-γ (LM2/1 + IFN-γ). Monocytes were then solubilized and analyzed as described in Materials and Methods. Cell lysates from stimulated A431 cells (A431) served as positive control. The upper panel shows probing of the nitrocellulose membranes with anti-phosphotyrosine antibodies (α-pTyr) and the arrow indicates the expression of p91 (STAT1). The lower panel shows a membrane reprobed with anti-STAT1 (α-STAT1). Data are representative of three similar experiments.
Figure 7
Figure 7
Production of IL-12 and IFN-γ in a murine model of septic shock is suppressed by treatment with CR3 antibodies. Pretreatment with 1 mg of antiCR3 (clone M1/70) markedly suppressed the serum levels of IL-12 p40 and p70 as well as serum levels of IFN-γ in BALB/c mice challenged by intravenous LPS (1 μg/mouse). Similar results were obtained with a different anti-CR3 antibody (clone 5C6). Data show the mean ± SD of groups consisting of three separately handled mice. *P <0.001, **P <0.0001, and ***P <0.05 versus the control (i.e., rat Ig treated) group as determined by the Student's t test. Data are representative of five experiments.
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