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A Role for CD154, the CD40 Ligand, in Granulomatous Inflammation - PubMed

A Role for CD154, the CD40 Ligand, in Granulomatous Inflammation

Julien Villeneuve et al. Mediators Inflamm. 2017.

Abstract

Granulomatous inflammation is a distinctive form of chronic inflammation in which predominant cells include macrophages, epithelioid cells, and multinucleated giant cells. Mechanisms regulating granulomatous inflammation remain ill-understood. CD154, the ligand of CD40, is a key mediator of inflammation. CD154 confers a proinflammatory phenotype to macrophages and controls several macrophagic functions. Here, we studied the contribution of CD154 in a mouse model of toxic liver injury with carbon tetrachloride and a model of absorbable suture graft. In both models, granulomas are triggered in response to endogenous persistent liver calcified necrotic lesions or by grafted sutures. CD154-deficient mice showed delayed clearance of carbon tetrachloride-induced liver calcified necrotic lesions and impaired progression of suture-induced granuloma. In vitro, CD154 stimulated phagocytosis of opsonized erythrocytes by macrophages, suggesting a potential mechanism for the altered granulomatous inflammation in CD154KO mice. These results suggest that CD154 may contribute to the natural history of granulomatous inflammation.

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Figures

Figure 1
Figure 1

Carbon tetrachloride liver injury progression in WT and CD154KO mice. (a) Relative levels of CD40 mRNA expression (arbitrary units) in the liver of WT and CD154KO mice at time 0 and after 3 and 9 intraperitoneal CCl4 injections (mean ± SD, n = 8). Representative HE-stained liver tissue sections of WT and CD154KO mice at time 0 (b) and after 1 (c), 2 (d), 3 (e), and 9 (f) intraperitoneal CCl4 injections (inj.). Top panels, low magnifications (scale bar 500 μm); bottom panels, high magnifications (scale bar 100 μm); (n = 8 at time 0, n = 4 at 1 injection, n = 4 at 2 injections, n = 8 at 3 injections, and n = 8 at 9 injections for each group of mice). (g) WT and CD154KO mice show similar hepatic cytolytic response after CCl4 administration. Serum measurements of albumin (gL), bilirubin (μmol/L), and liver enzymes (IU/L) after 1 CCl4 injection in WT and CD154KO mice (mean ± SD, n = 5).

Figure 2
Figure 2

Differential clearance of dystrophic calcified necrotic liver lesions in WT and CD154KO mice: von Kossa staining. Representative von Kossa-stained liver tissue sections of WT and CD154KO mice at time 0 (a) and after 3 (b and c) and 9 (d) CCl4 injections (inj.). Top panels, low magnifications (scale bar 500 μm); bottom panels, high magnifications (scale bar 100 μm); (n = 8 for each condition in each group of mice). (c) High magnifications (scale bar 50 μm) from (b) (T = 3 injections), highlighting MGCs associated to dystrophic calcified areas (arrows).

Figure 3
Figure 3

Immunohistochemical staining of monocyte/macrophage cells and cytokine/chemokine expression in WT and CD154KO mice after CCl4 injections. (a) Representative liver tissue sections from WT and CD154KO mice stained with the anti-F4/80 antigen monoclonal antibody after 3 and 9 CCl4 injections (inj.). Top panels, low magnifications (scale bar 500 μm); bottom panels, high magnifications (scale bar 100 μm) (n = 8 for each condition in each group of mice). Relative liver levels of F4/80 (b), IL-4 (c), MCP-1 (d), IL-6 (e), and MIP-2 (f) mRNA (fold arbitrary units), in WT and CD154KO mice at time 0 and after 3 and 9 CCl4 injections (inj.) (mean ± SD, ∗p < 0.05, n = 8).

Figure 4
Figure 4

Absorbable suture bundles undergo a rapid bioresorption between weeks 8 and 10 postimplantation. (a) Tissue sections (low and high magnifications, scale bars 500 and 50 μm for left and right panels, resp.) were obtained at week intervals and stained with Masson's trichrome. Microphotographs shown correspond to a representative kinetic of 3 experiments performed. Bundle contours showing rapid shrinking by week 8 are indicated by arrowheads. Red arrows indicate the nonabsorbable thread which, upon section by the microtome, creates an artefactual void on the tissue section (asterisk); (b) quantification of (a) suture bundle bioresorption.

Figure 5
Figure 5

Differential clearance of suture bundles in WT and CD154KO mice. Representative Masson's trichrome-stained tissue sections from WT and CD154KO mice at week 10. (a and d) Low magnification (scale bar 500 μm); arrows indicate the nonresorbable thread which, upon section by the microtome, creates an artefactual void on the tissue section (asterisk); squares highlight the FBR in subcutaneous tissues. (b and e) High magnifications (scale bar 20 μm); the granulomas are composed of macrophages and multinucleated giant cells (MGCs) (arrows) bordered by fibrosis (green/blue). (c and f) Representative liver tissue sections from WT and CD154KO mice stained with the anti-F4/80 antigen monoclonal antibody at week 10; high magnifications (scale bar 20 μm), macrophages, and MGCs (arrows) express the F4/80 antigen macrophagic marker. (g) Quantification of granuloma areas in WT and CD154KO mice (∗p < 0.05).

Figure 6
Figure 6

CD154 enhances phagocytosis by in vitro-derived macrophages. (a and b) CD40 is expressed by in vitro-derived macrophages and multinucleated giant cells (MGCs). (a) Macrophages derived from peripheral blood monocytes were immunostained for CD40 expression. Top panel, representative microphotographies of in vitro-derived macrophages (×100 and ×200 magnifications); bottom panel, CD40 immunostaining; nuclei were counterstained with DAPI (×400 magnification). (b) MGCs were obtained by IL-4-induced macrophage fusion and immunostained for CD40 expression. Top panel, representative microphotographies of in vitro-derived MGCs (×100 and ×200 magnifications); bottom panel, CD40 immunostaining; nuclei were counterstained with DAPI (×400 magnification). (c and d) CD154 enhances phagocytosis by in vitro-derived macrophages. (c) Cultures of in vitro-derived macrophages were assayed for phagocytosis in the presence or not of rsCD154. (d) Results obtained after fusion induction by IL-4. Cells were prestimulated 24 hours with 200 ng/mL rsCD154, and phagocytosis of opsonized erythrocytes was measured at various time-points (mean ± SD, n = 6; ∗p < 0.05).

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