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. 1997 Mar 4;94(5):1931-6.
doi: 10.1073/pnas.94.5.1931.

Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis

F Mach  1 , U Schönbeck, G K Sukhova, T Bourcier, J Y Bonnefoy, J S Pober, P Libby
Affiliations

Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis

F Mach et al. Proc Natl Acad Sci U S A. .

Abstract

Increasing evidence supports involvement of inflammation and immunity in atherogenesis. We report here that CD40 ligand (CD40L), an immunoregulatory signaling molecule heretofore considered largely restricted to recently activated CD4+ T lymphocytes, is expressed by human vascular endothelial cells (EC), smooth muscle cells (SMC), and human macrophages in vitro, and is coexpressed with its receptor CD40 on all three cells types in human atherosclerotic lesions in situ. Cultured human vascular EC, SMC, and human macrophages all constitutively expressed CD40L mRNA as well as protein. Stimulation with interleukin 1beta, tumor necrosis factor alpha, or interferon gamma increased surface levels and de novo synthesis of CD40L on all three cell types. CD40L expressed on EC, SMC, and macrophages exhibited biological activity, as it induced B7.2 expression on B cells. Human vascular SMC also constitutively expressed CD40, the receptor for CD40L, and through CD40 signaling, human recombinant CD40L induced expression of proinflammatory cytokines in these cells, identifying SMC as a target for CD40L. Human atherosclerotic lesions (n = 8) showed expression of immunoreactive CD40L on EC, SMC, and macrophages, while normal arterial tissues (n = 5) contained no CD40L. In atheroma CD40L+ cells often also expressed CD40. These observations establish human vascular EC, SMC, and human macrophages as a novel source of CD40L, and point to T cell-independent CD40 signaling, and a broader function of this pathway in regulation of nonimmune cells, as illustrated here by potential autocrine and paracrine activation during atherogenesis.

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Figures

Figure 1
Figure 1
Human vascular SMC, EC, and human macrophages express CD40L and CD40 in vitro. (A) PCR with reverse transcribed total RNA preparations (+) obtained from human vascular SMC, EC, or human macrophages (MØ) resulted in the detection of the CD40L–cDNA product of the expected size (520 bp). Genomic contamination was excluded by PCR of nonreverse transcribed mRNA preparations (−), and cDNA derived from phorbol 12-myristate 13-acetate (PMA)-stimulated (50 ng/ml, 24 h) CD4+ T cells (CD4+) was used as a positive control. (B) Human vascular SMC, EC, or human MØ express CD40L as a cell-associated 33-kDa protein. Cell preparations of unstimulated SMC, EC, and MØ were analyzed by Western blot. Preparations of PMA-stimulated (50 ng/ml, 24 h) CD4+ T cells (CD4+) were used as positive control. Preincubation of anti-CD40L antibody with human rCD40L (1 μg/ml) inhibited detection of CD40L in cell preparations, demonstrating the specificity of the antibodies. (C) De novo synthesis of CD40L is demonstrated by radioimmunoprecipitation analysis of unstimulated [insulin/transferrin (IT); ref. 24] as well as human recombinant IL-1β (10 ng/ml), TNF-α (50 ng/ml), or IFN-γ (1000 units/ml) treated (24 h) SMC, cultured under serum-free conditions. (D) De novo synthesis of CD40 on SMC is demonstrated by radioimmunoprecipitation analysis of unstimulated (IT) as well as human recombinant IL-1β (10 ng/ml), TNF-α (50 ng/ml), or IFN-γ (1000 units/ml) treated (24 h) cells, cultured under serum-free conditions. Results shown were reproduced in four independent experiments.
Figure 2
Figure 2
FACS analysis of human vascular SMC, EC, and human macrophages (MØ) cultured in serum-containing medium, stained for CD40L (solid histograms), as well as isotype control (open histograms) are shown. Each panel is a histogram representing cell numbers (y axis) vs. log fluorescence intensity (x axis) for 30,000 viable cells. Results shown were reproduced in three independent experiments.
Figure 3
Figure 3
Induction of proinflammatory cytokine expression in human vascular SMC, EC, or human macrophages by human rCD40L. (A) Supernatants of rCD40L-stimulated (5 μg/ml; •) or unstimulated (○) human vascular SMC, EC, or human macrophages (MØ) were analyzed for IL-6 and IL-8 by ELISA. (B) Macrophages were incubated with rCD40L (5 μg/ml) in the absence (•) or presence (○) of anti-CD40L mAb and analyzed for IL-1β and TNF-α release. Error bars represent SD. Human rCD40L significantly increased cytokine expression relative to control [without (w/o) rCD40L or with anti-CD40L, respectively] after 24 h (P ≤ 0.05, Student’s unpaired t test). Results shown were reproduced for each of the cytokines in three independent experiments.
Figure 4
Figure 4
Expression of CD40L and CD40 in human vascular atherosclerotic lesions in situ. (A and B) Low power views (×40) of frozen sections of human carotid lesions show expression of CD40L and CD40 in the shoulder region of the plaque. (C–F) High power views (×400) of this region revealed specific staining for CD40L and CD40 on SMC and macrophages (C and D), as well as on EC of the luminal border (E and F). As demonstrated here for CD40L on EC (G and H), cell types were characterized by immunofluorescence-double staining as described. The lumen of the artery is at the top of each photomicrograph. Analysis of eight atheroma showed similar results.
Figure 5
Figure 5
Coexpression of CD40L and CD40 in human vascular atherosclerotic lesions in situ. (A) Low power view (×40) of a human carotid atherosclerotic plaque, stained for macrophages (MØ) (CD68). The rectangle indicates the macrophage-rich region sampled in the high power views (×400, B and C) of an adjacent section to the one depicted in A, visualized with different emission filters. (B) Fluorescent staining for CD40L (red) and nuclear DNA (blue). (C) Immunofluorescent staining for CD40 (green) in the same section shown in B. The lumen of the artery is at the top of each photomicrograph. Analysis of four atheroma showed similar results.
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