The role of platelet factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury

Abstract

The robust inflammatory response that occurs during ischemia reperfusion (IR) injury recruits factors from both the innate and adaptive immune systems. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR injury has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration were decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissues. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after IR at both the mRNA and protein levels. In conclusion, these findings demonstrate that may PF4 represent an important mediator of local and remote tissue damage.

Figure 1. PF4 deposition increased dramatically in the intestine and lung of B6 mice after mesenteric IR injury.

Tissue sections of lung (A-C) and intestine (D-F) from B6 after 30 minutes of mesenteric ischemia and 3 hrs of reperfusion were stained for PF4 (red) and counterstained with hematoxylin (blue). Images are representative of 3–4 mice per group. (G) PF4 mRNA levels in lung. (H) PF4 mRNA levels in intestinal epithelial cells. All images shown are 200× and 400× magnification. *p≤0.05, **p≤0.01, for IR compared to sham controls.

Figure 2. Intestinal and lung injury is reduced after mesenteric ischemia/reperfusion in PF4^-/- mice:

Hematoxylin and eosin stained sections of mouse small intestine and lung after 30 minutes of ischemia and 3 hours reperfusion. A total of 5–8 mice were used for each control and experimental groups in two experiments. (A-D) Images of lung from sham and IR (E-H). Images of intestinal villi from sham and IR. All images shown are 200× magnification. (I,J) Injury score (mean ± SD) in lung and intestine.(K) PF4 plasma levels in B6 and PF4-/- mice before and after mesenteric IR injury. *p≤0.05, **p≤0.01, and ***p≤0.001 for IR compared to sham controls.

Figure 3. Neutrophil (PMN) and monocyte infiltration is reduced in the lung of PF4-/- mice after mesenteric IR injury.

Tissue sections of lung of B6 and PF4-/- mice after 30 minutes of mesenteric ischemia and 3 hrs of reperfusion and were stained for neutrophils (A-F, red) and monocytes (G-L, red) and counterstained with hematoxylin (blue). A total of 5–8 mice were used for each control and experimental groups in two experiments. (M) Neutrophil infiltration score, (N) Monocyte infiltration score. ns: not significant *p≤0.05 for IR compared to sham controls. Red: Positive Staining.

Figure 4. Tissue damage in PF4-/- mice is not associated with complement deposition.

Tissue sections of lung (A-F) and intestine (G-L) from B6 and PF4-/- mice after 30 minutes of mesenteric ischemia and 3 hrs of reperfusion and were stained C3 complement factor (red) and counterstained with hematoxylin (blue). Images are representative of 3–4 mice per group.

Figure 5. Tissue damage in PF4-/- mice is not associated with immunoglobulin (Ig) deposition.

Tissue sections of lung and intestine from B6 and PF4-/- mice after 30 minutes of mesenteric ischemia and 3 hrs of reperfusion were stained for IgM (A-L, red) and IgA (M-X, red) and counterstained with hematoxylin (blue). Images are representative of 3–4 mice per group. Red: Positive Staining.

Figure 6. Immunoglobulin (Ig) plasma levels in B6 and PF4-/- mice before and after mesenteric IR injury.

Plasma samples obtained from B6 and PF4-/- mice before and after mesenteric IR injury were subjected to multiplex analysis. (A) IgM plasma levels. (B) IgA plasma levels, (C) IgG plasma levels. ns: not significant *p≤0.05, **p≤0.01, and ***p≤0.001 for IR compared to sham controls. A total of 5–8 mice were used for each control and experimental groups in two experiments.

Figure 7. Transfusion of PF4-/- platelets into platelet depleted B6 mice fails to restore local and remote tissue damage after mesenteric IR injury.

Hematoxylin and eosin stained lung and small intestine sections of PF4-/- mice transfused with B6 platelets and vise versa after 30 minutes of ischemia and 3 hours reperfusion. A total of 5–8 mice were used for each control and experimental groups in a total of two experiments. (A-F) Images of lung from sham and IR. (H-M) Images of small intestine from sham and IR. All images shown are 200× magnification. (G,N) Injury score (mean ± SD) in lung and small intestine respectively. *p≤0.05 and ***p≤0.001 for IR compared to sham controls.