Platelet-associated CD40/CD154 mediates remote tissue damage after mesenteric ischemia/reperfusion injury

Abstract

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage.

Figure 1. Intestinal and lung injury is reduced after mesenteric ischemia/reperfusion in CD40^−/− and CD154^−/− mice.

Hematoxylin and eosin stained sections of mouse small intestine after 30 minutes of ischemia and 3 hours reperfusion. Images are representative of 3–4 mice per group in two experiments. (A–F) Images of intestinal villi from sham and I/R. (G–L) Images of lung from sham and I/R, platelet-deficient sham and I/R. All images shown are 200×magnification. (M, N) Injury score (mean ± SD) in intestine and lung. *p≤0.05, **p≤0.01, and ***p≤0.001 for I/R compared to sham controls.

Figure 2. Platelets numbers are not decreased in CD40^−/− and CD154^−/− mice after mesenteric ischemia/reperfusion.

CD40^−/− and CD154^−/− mice were bled before and after mesenteric I/R via cardiac puncture. Platelet numbers were determined using Hemavet 850 (Drew Scientific, Farmington, CT).

Figure 3. Transfusion of either CD40^−/− and CD154^−/− platelets protects platelet depleted B6 mice from remote lung injury but not from local intestinal injury.

(A–L) Hematoxylin and eosin stained sections of mouse small intestine and lung from B6 and platelet depleted B6 mice transfused with either CD40^−/− or CD154^−/− platelets after 30 minutes of ischemia and 3 hours reperfusion. Images are representative of 3–4 mice per group in two experiments. All images shown are 200×magnification. (M)Intestinal injury score and (N) Lung injury score (mean ± SD). ns: not significant *p≤0.05, **p≤0.01, and ***p≤0.001 for I/R compared to sham controls.

Figure 4. Reduced platelet and complement deposition in lungs after ischemia/reperfusion in CD40^−/− and CD154^−/− platelet transfused B6 mice.

(A–R) Tissue sections of lung from B6 (top row) and platelet depleted B6 mice transfused with either CD154^−/− (bottom row) or CD40^−/− (middle row) platelets and mice after 30 minutes of mesenteric ischemia 3 hrs of reperfusion and sham controls were stained for platelets (A–I) or C3 complement factor (J–R) and counterstained with hematoxylin (blue). Images are representative of 3–4 mice per group. Large arrows: indicate areas with increased intensity of staining. Small arrows: indicate areas with reduced intensity of staining.

Figure 5. Remote lung injury is re-established in CD154^−/− and CD40^−/− mice transfused with B6 platelets.

(A–D) Hematoxylin and eosin, (E–H) platelets and (I–L) C3 complement factor stained sections of mouse lung from CD154^−/− and CD40^−/− mice transfused with B6 platelets after 30 minutes of ischemia and 3 hours reperfusion. Images are representative of 3–4 mice per group in two experiments. All images shown are 200×magnification. (M)Injury score (mean ± SD) in intestine and lung. ns: not significant *p≤0.05, **p≤0.01, and ***p≤0.001 for I/R compared to sham controls. Small arrows: indicate areas of positive staining.