Enhanced susceptibility to endotoxic shock and impaired STAT3 signaling in CD31-deficient mice - PubMed (original) (raw)

Enhanced susceptibility to endotoxic shock and impaired STAT3 signaling in CD31-deficient mice

Michael Carrithers et al. Am J Pathol. 2005 Jan.

Abstract

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), an adhesion molecule expressed on hematopoietic and endothelial cells, mediates apoptosis, cell proliferation, and migration and maintains endothelial integrity in addition to its roles as a modulator of lymphocyte and platelet signaling and facilitator of neutrophil transmigration. Recent data suggest that CD31 functions as a scaffolding protein to regulate phosphorylation of the signal transducers and activators of transcription (STAT) family of signaling molecules, particularly STAT3 and STAT5. STAT3 regulates the acute phase response to innate immune stimuli such as lipopolysaccharide (LPS) and promotes recovery from LPS-induced septic shock. Here we demonstrate that CD31-deficient mice have reduced survival during endotoxic LPS-induced shock. As compared to wild-type controls, CD31-deficient mice showed enhanced vascular permeability; increased apoptotic cell death in liver, kidney, and spleen; and elevated levels of serum tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFNgamma), MCP-1, MCP-5, sTNRF, and IL-6. In response to LPS in vivo and in vitro, splenocytes and endothelial cells from knockout mice had reduced levels of phosphorylated STAT3. These results suggest that CD31 is necessary for maintenance of endothelial integrity and prevention of apoptosis during septic shock and for STAT3-mediated acute phase responses that promote survival during septic shock.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Decreased survival in CD31-deficient mice treated with LPS. Kaplan-Meyer curves demonstrate 100% survival of wild-type mice at two doses of LPS (E. coli serotype 055:B5), 200 μg (A) and 600 μg (B) (n = 8 for each dose). In contrast, CD31-deficient mice had 43% survival at 200 μg (n = 7) and 0% survival at 600 μg (n = 8).

Figure 2

Figure 2

Pulmonary edema and congestion occur in CD31-deficient mice following LPS stimulation. Representative low- (A–C) and high- (D–F) power H&E micrographs of lungs harvested from age- and sex-matched wild-type control mice (WT), wild-type mice treated with LPS (WT + LPS), and CD31-deficient mice treated with LPS (CD31 + LPS). Lung tissue harvested from CD31-deficient mice (not shown) appeared indistinguishable from WT control mice (A and D). Treatment of WT mice with LPS resulted in no appreciable changes in lung morphology (compare A and D with B and E). In contrast, treatment of CD31-deficient mice with LPS resulted in significant vascular congestion and edema (Table 1 and C and F). Bar, 100 μm (A to C); bar, 50 μm (D to F.)

Figure 3

Figure 3

Renal vascular congestion and endothelial cell apoptosis occur in CD31-deficient mice following LPS stimulation. Representative low- (A–C) and high- (D–O) power H&E micrographs of kidneys harvested from age- and sex-matched wild-type control mice (WT), wild-type mice treated with LPS (WT + LPS), and CD31-deficient mice treated with LPS (KO + LPS). Kidney tissue harvested from CD31-deficient mice (not shown) appeared indistinguishable from WT control mice (A, D, E, J, and K). Treatment of WT mice with LPS resulted in modest glomerular and peri-tubular capillary congestion (compare D, E, J, and K with F, G, L, and M). In contrast, treatment of CD31-deficient mice with LPS resulted in significant glomerular and peri-tubular capillary vascular congestion and pyknosis of peri-tubular capillary endothelial cell nuclei (arrow) (H, I, N, and O). Bar, 100 μm (A–C); bar, 50 μm (D–I); bar, 10 μm (J to O).

Figure 4

Figure 4

Renal peri-tubular capillary endothelial cell apoptosis occurs in CD31-deficient mice following LPS stimulation. Representative low- (A, C, and E) and high- (B, D, and F) power micrographs TUNEL-stained sections of kidneys harvested from age- and sex-matched wild-type control mice (WT), wild-type mice treated with LPS (WT + LPS), and CD31-deficient mice treated with LPS (KO + LPS). Kidney tissue harvested from CD31-deficient mice (not shown) appeared indistinguishable from WT control mice having no TUNEL positivity (A and B). Treatment of WT mice with LPS yielded no TUNEL positivity (compare A and B with C and D). In contrast, treatment of CD31-deficient mice with LPS resulted in significant TUNEL positivity of peri-tubular capillary endothelial cell nuclei (E and F). Bar, 100 μm (A, C, and E); bar, 50 μm (B, D, and F).

Figure 5

Figure 5

Hepatic vascular congestion and sinusoidal neutrophilic infiltration and apoptosis occur in CD31-deficient mice following LPS stimulation. Representative low- (A to C) and high- (D to F) power H&E micrographs of livers harvested from age- and sex-matched wild-type control mice (WT), wild-type mice treated with LPS (WT + LPS), and CD31-deficient mice treated with LPS (CD31 + LPS). Liver tissue harvested from CD31-deficient mice (not shown) appeared indistinguishable from WT control mice (A, D, and G). Treatment of WT mice with LPS resulted in very modest hepatic vascular and sinusoidal congestion (compare A, D, and G with B, E, and H). In contrast, treatment of CD31-deficient mice with LPS resulted in significant hepatic vascular and sinusoidal congestion, sinusoidal neutrophilic infiltration (C and F), and portal monocytic and neutrophilic infiltration (I and J) with occasional neutrophilic apoptosis (J, circle). Bar, 100 μm (A–C); bar, 50 μm (D–F); bar, 50 μm (G–J).

Figure 6

Figure 6

Splenic congestion and apoptosis occurs in CD31-deficient mice following LPS stimulation. A and B: Representative low-power micrographs of spleens from age- and sex-matched wild-type (WT) and CD31-deficient mice (KO + LPS) treated with LPS. LPS treatment elicited significantly more congestion in the red pulp of spleens of the KO mice. Bar, 100 μm. C and D: Peroxidase-labeled micrographs of sections of splenic tissue from WT (C) and CD31 KO (D) animals stained with TUNEL reagent. A robust increase in TUNEL labeling was noted in CD31 KO animals following treatment with LPS while no appreciable increase in TUNEL labeling was noted in WT animals. Bar, 50 μm (representative of four experiments).

Figure 7

Figure 7

LPS treatment elicits significant increases in TNFα and other selected cytokine serum levels in CD31-deficient mice compared to WT mice. A: ELISA assays showed approximately a 10-fold increase in TNFα levels in the sera of CD31-deficient mice (233 pg/ml) compared to WT mice (25 pg/ml), n = 8, P < 0.003. B: Higher levels of several cytokines including INFγ, IL-6, MCP-1, MCP-5, and sTNFR1 were noted when sera was assayed using a Panomics cytokine array assay to assess cytokine profiles over a 24-hour time period of WT and CD31 KO animals treated with LPS. Other cytokines (VEGF [shown] and TPO, TARC, SCF, RANTES, MIP-3B, MIP-2, MIP-1A, LEPTIN, KC, IL-9, IL-5, IL-4, IL-3, IL-2, IL-17, IL-13, IL-12, IL-10, GMCSF, GCSF, EOTAXIN, CTACK, and 6CKINE [not shown]) similarly assessed did not exhibit any appreciable changes in response to LPS. Data were accrued from three independent experiments, each run in duplicate. Results were confirmed using the BD cytodex bead assay (not shown). A representative series of cytokine arrays illustrating the differences between the sera harvested from WT and CD31 KO animals following LPS treatment is included.

Figure 8

Figure 8

Splenic lysates and splenic lymphocytes harvested from CD31-deficient mice exhibit decreased pSTAT3 levels compared to WT mice following LPS stimulation. A: The top panel represents quantitation of the relative pSTAT3 levels (sex- and age-matched averages of five and five CD31 KO animals treated with LPS and harvested 24 hours later) of splenic tissue harvested from wild-type (WT) and CD-31-deficient (CD31 KO) animals, normalized to total STAT3 (P < 0.004). The KO spleens exhibit a 75% decrease in pSTAT3 compared to WT spleens. The bottom panel is a representative Western blot of pSTAT3 and STAT3 from one WT and one CD31 KO animal of the group tested. B: The top panel represents the quantitation of the relative pSTAT3 levels of lymphocytes harvested from the spleens of WT and KO mice and cultured in vitro for 24 hours in the absence and presence of 10 and 100 ng/ml LPS. The lymphocytes harvested from CD31 KO spleens exhibit a significant decrease in pSTAT3 compared to WT splenic lymphocytes in the absence of LPS and at both LPS concentrations, normalized to total STAT3. The bottom panel is a Western blot of pSTAT3 and STAT3 from the lymphocytes harvested from a WT and a CD31 KO animal. C–F: Immunofluorescence micrographs of 6-μm frozen sections of splenic tissue from WT (C) and CD31-deficient (KO) (D) animals stained with pSTAT3 (red fluorescence) and DAPI (blue fluorescence). A 3.7-fold increase (P < 0.04) in pSTAT3 labeling was noted in WT animals following treatment with LPS (compare C and D) while no appreciable increase in pSTAT3 labeling was noted in CD31 KO animals (0.95, P = 0.45) (compare E and F) (representative of three experiments). G-J: Immunofluorescence micrographs of 6-μm frozen sections of splenic tissue from WT (G and I) and CD31-deficient (KO) (H and J) animals stained with pSTAT3 (red fluorescence) and Mac-3 (green fluorescence) revealed essentially no differences in macrophage pSTAT3 localization before (G and H) or following (I and J) LPS treatment. The increased pSTAT3 nuclear expression noted in the WT LPS sample (I) is manifested as red to yellow fluorescence due to the intensity of the fluorescence. Essentially no nuclear pSTAT3 localization was appreciated in Mac-3-positive cells as evidenced by the lack of any pSTAT3 nuclear staining in the cells expressing green fluorescence in their cytoplasm (insets).

Figure 9

Figure 9

Immortalized pulmonary endothelial cells derived from CD31-deficient mice exhibit decreased levels of p-STAT3 in response to LPS exposure in vitro. A: Top, representative Western blot of KO lung endothelial cells revealed decreased levels of pSTAT3 compared to PECAM-1-reconstituted cells (RC), although both cell types expressed approximately equal amounts of total STAT3. Following stimulation with 100 ng/ml LPS, unlike the increases noted in the RC cells, the KO cells exhibited a significantly blunted pSTAT3 response. Bottom, average of five independent experiments illustrating the blunted pSTAT3 response in KO cells (*, P < 0.0 5). B: Western blot analysis of KO lung endothelial cells and PECAM-1-reconstituted cells stimulated with 100 ng/ml LPS over a 12-hour time period revealed decreased levels of pSTAT3 in KO endothelial cells compared to PECAM-1-reconstituted (RC) endothelial cells, although both cell types expressed approximately equal amounts of total STAT3 (n = 5; *, P value < 0.05). C to F: Immortalized pulmonary endothelial cells derived from CD31-deficient mice exhibit decreased nuclear localization of pSTAT3 in response to LPS exposure in vitro. Immunofluorescence microscopy of cultured PECAM-1-reconstituted endothelial cells (RC) (C and E) and KO endothelial cells (D and F) in the absence (C and D) and presence (E and F) of LPS revealed no appreciable nuclear localization of pSTAT3 in both cell types in the absence of LPS (C and D). In contrast, the presence of nuclear pSTAT3 was noted in RC cells but was significantly reduced in KO cells following LPS treatment (E and F). Bar, 50 μm.

Figure 10

Figure 10

IL-6 stimulation of PECAM-1 expressing splenic lymphocytes elicits increased expression of pSTAT3 and sodium orthovanadate inhibition of phosphatase activity in IL-6 stimulated splenic lymphocytes elicits increased phosphorylation of STAT3 in CD31-deficient cultures. WT and KO splenic lymphocyte cultures were stimulated with IL-6 (1 ng/ml) for 15 to 30 minutes in the absence (WT and KO lymphocytes) and presence of Sodium orthovanadate (WT and KO lymphocytes + NaOV). In the absence of sodium orthovanadate, Western blot analyses of lysates revealed marked decreases in STAT3 phosphorylation in the IL-6 stimulated CD31-deficient lymphocyte cultures (open bars) compared to wild-type cultures (shaded bars). In the presence of sodium orthovanadate, the levels of pSTAT3 in the IL-6 stimulated CD31-deficient lymphocyte cultures were found to be similar to the levels observed in similarly treated wild-type cultures (averages of two independent experiments).

Figure 11

Figure 11

TLR4 expression of WT and CD31-deficient splenocytes and endothelial cells remains constant and does not change following LPS treatment. Representative FACS analyses of WT (A) and KO (B) splenocytes in the absence and presence of LPS; KO (C) and PECAM-1 reconstituted (D) lung endothelial cells and KO (E) and WT (F) brain endothelial cells illustrating essentially no differences in TLR4 expression. This was confirmed by Western blot analyses (data not shown).

Figure 12

Figure 12

Current working model of the role of PECAM-1 as a modulator of cytokine expression. PECAM-1 facilitates down-regulation of LPS-induced IL-6-mediated cytokine induction by modulating the localization and activities of SHP-2 (WT). In the absence of PECAM-1, SHP-2 binds and dephosphorylates STAT3 rendering it unable to translocate to the nucleus and initiate the down-regulation of cytokine expression, which may potentiate sustained cytokine expression in CD31 KO animals.

References

    1. Newman P, Berndt MC, Gorski J, White GC, Lyman S, Paddock C, Muller WA. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene family. Science. 1990;247:1219–1222. - PubMed
    1. Muller W, Ratti C, McDonnell S, Cohn Z. A human endothelial cell-restricted, externally disposed plasmolemmal protein enriched in intercellular junctions. J Exp Med. 1989;170:399–414. - PMC - PubMed
    1. Abelda S, Oliver P, Romer L, Buck C. EndoCAM: a novel endothelial cell-cell adhesion molecule. J Cell Biol. 1990;110:1227–1237. - PMC - PubMed
    1. Ohto H, Maeda H, Shibata Y, Chen RF, Ozaki Y, Higashihara M, Takeuchi A, Tohyama H. A novel leukocyte differentiation antigen: two monoclonal antibodies TM2 and TM3 define a 120-kd molecule present on neutrophils, monocytes, platelets, and activated lymphoblasts. Blood. 1985;66:873–881. - PubMed
    1. Stockinger H, Gadd SJ, Eher R, Majdic O, Schreiber W, Kasinrerk W, Strass B, Schnabl E, Knapp W. Molecular characterization and functional analysis of the leukocyte surface protein CD31. J Immunol. 1990;145:3889–3897. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources