Hemopexin alleviates sterile inflammation in ischemia-reperfusion-induced lung injury

Abstract

Introduction: Pulmonary ischemia-reperfusion (IR) injury (IRI) plays a significant role in various lung disorders and is a key factor in the development of primary graft dysfunction following lung transplantation. Hemopexin (Hx) is the major serum scavenger protein for heme, which is a prooxidant and pro-inflammatory compound. In the current study, we hypothesized that Hx could confer beneficial effects in sterile inflammation induced by IR-mediated lung injury.

Methods: To examine this hypothesis, we administered Hx in an experimental mouse model of unilateral lung IRI.

Results: Our results demonstrate that treatment with Hx alleviated histopathological signs of inflammation in ischemic lungs, as evidenced by a reduction in the number of infiltrating neutrophils and decreased levels of perivascular edema. In addition, thrombotic vaso-occlusion in pulmonary blood vessels of IRI lungs was reduced by Hx. Immunohistochemical analysis revealed that Hx inhibited the up-regulation of heme oxygenase-1, an enzyme highly induced by heme, in ischemic lungs. Finally, Hx administration caused a decrease in the levels of circulating B- and CD8+ T-lymphocytes in the peripheral blood of mice with pulmonary IRI.

Conclusion: These findings suggest that the serum heme scavenger protein Hx holds therapeutic promise in alleviating lung IRI-mediated sterile inflammation. Thus, Hx may represent a preemptive therapeutic approach in IR-related lung disorders such as primary graft dysfunction in lung transplantation.

Keywords: heme; hemopexin; ischemia-reperfusion injury; lung; sterile inflammation.

Figure 1

Mouse model of unilateral lung IRI. A model of unilateral pulmonary IRI was established in C57BL6/J mice by unilateral pulmonary clamping of the left lung hilum for 1.5h after left-sided thoracotomy followed by 4 h of reperfusion.

Figure 2

Comparing lung injury scores between ischemic and control lungs in the unilateral pulmonary IRI mouse model - treatment with Hx. Lung tissue damage in the applied mouse model of unilateral pulmonary IRI was evaluated according to the lung injury score by Murakami et al. (22). In this scoring system, lung injury was graded on a scale of 0 to 4 (0, normal; 1, light; 2, moderate; 3, strong; 4 intense) for congestion, edema, infiltration, and hemorrhage in multiple areas (10 microscopic view fields). The left ischemic lung (IRI) exhibited significantly higher levels of lung injury than the right control lung (Con) (IRI vs. Con: 9 (6) vs. 2 (2), respectively: p = 0.02, IRI vs. Sham: 9 (6) vs. 0 (0), respectively: p = 0.008). Moreover, in the group of mice treated with Hx (IRI group vs. Hx treated group: 9 (6) vs. 5 (1.5), respectively: p = 0.037). (n=5 mice in each group). N.S., not significant.

Figure 3

Hx reduces number of infiltrating neutrophils in the ischemic lung of the unilateral pulmonary IRI mouse model. Infiltration of neutrophils was histologically determined by PAS staining, as detailed in Materials and Methods. The indicated red-dyed cells (yellow arrows) are neutrophils. Numbers of neutrophils were significantly higher in the left ischemic compared to the right control lung (36.3 (33.5) vs. 14.8 (10.1), respectively: p = 0.047). In the group of Hx-treated mice significantly lower numbers of neutrophils were determined in the left ischemic lung (IRI group vs. Hx treated group: 36.3 (33.5) vs. 16.3 (27.2), respectively: p = 0.047). n=6 mice for the vehicle group (IRI and Con), and, n=6 mice for the Hx-treated group (IRI+Hx and Con+Hx). N.S., not significant.

Figure 4

Hx alleviates perivascular edema in ischemic lungs of the unilateral pulmonary IRI mouse model. (A) Levels of perivascular edema were evaluated histologically by assessing five vessels of right and left lungs, respectively. Distances from tunica intima to tunica intima (Dv) were compared with the shortest diameter from tunica adventia to tunica adventia (Dc). Shortest distances were utilized as measured values to compensate for distortion of the results caused by different cutting planes. To compare different vessel sizes, the results were combined into one parameter with the formula cuff rea/vessel area = D_c ²-D_V ²/D_V ², as indicated. (B) The extent of perivascular edema, as determined by the cuff area-to-vessel area ratio, was markedly higher in the left ischemic compared to the right control lung (10.6 (5.7) vs. 6.5 3.4), respectively: p = 0.031). Increased levels of cuff area-to-vessel area ratio in ischemic lungs were reduced by treatment with Hx (IRI group vs. Hx treated group: 10.6 (5.7) vs. 5.9 (2.7), respectively: p = 0.032). n=5 mice for the vehicle group (IRI and Con), and, n=5 mice for the Hx-treated group (IRI+Hx and Con+Hx). (Outlier [35.4] in the IRI with vehicle group was excluded). N.S., not significant.

Figure 5

Hx alleviates intravascular thrombus formation in the ischemic lung of the unilateral pulmonary IRI mouse model. Histological studies revealed significantly higher numbers of thrombotic blood vessels in the left ischemic compared to the right control lung (72.5 (15.6)% vs. 0 (8.1)%, respectively: p = 0.008). The number of thrombotic pulmonary vessels was significantly reduced in the group of mice treated with Hx (IRI group vs. Hx treated group: 72.5 (15.6)% vs. 34.7 (33.7)%, respectively: p = 0.008). n=5 mice for each group. N.S., not significant.

Figure 6

Hx blocks up-regulation of HO-1 expression in the ischemic lung of the unilateral pulmonary IRI mouse model. Immunohistochemical studies revealed significantly higher levels of HO-1 expression in the left ischemic relative to the right control lung (0.68 (0.29) vs. 0.21 (0.72), respectively: p = 0.005). In the group of Hx-treated mice, HO-1 expression levels were significantly reduced in the ischemic left lung (IRI group vs. Hx treated group: 0.68 (0.29) vs. 0.29 (0.42), respectively: p = 0.049). n=10 (5 lungs/mice, two measurements for each lung in each group. N.S., not significant.

Figure 7

Hx affects levels of immune cells in peripheral blood from mice with unilateral lung IRI. The gating strategy for flow cytometry analysis of various immune cells is described in Materials and Methods. (A, B) Significant lower levels of B- (CD3⁺CD19⁺) and CD8+ T (CD3⁺CD8⁺) cells were detected in IRI mice with Hx compared to mice without Hx treatment (n = 5 mice in each group: p=0.037 and 0.008, respectively). (C) Significantly higher levels of dendritic cells were found in IRI mice with Hx treatment than mice without Hx treatment (n = 5, p=0.04).