The systemic inflammatory response after spinal cord injury in the rat is decreased by α4β1 integrin blockade

Abstract

Abstract The systemic inflammatory response syndrome (SIRS) follows spinal cord injury (SCI) and causes damage to the lungs, kidney, and liver due to an influx of inflammatory cells from the circulation. After SCI in rats, the SIRS develops within 12 h and is sustained for at least 3 days. We have previously shown that blockade of CD11d/CD18 integrin reduces inflammation-driven secondary damage to the spinal cord. This treatment reduces the SIRS after SCI. In another study we found that blockade of α4β1 integrin limited secondary cord damage more effectively than blockade of CD11d/CD18. Therefore we considered it important to assess the effects of anti-α4β1 treatment on the SIRS in the lung, kidney, and liver after SCI. An anti-α4 antibody was given IV at 2 h after SCI at the fourth thoracic segment and the effects on the organs were evaluated at 24 h post-injury. The migration of neutrophils into the lungs and liver was markedly reduced and all three organs contained fewer macrophages. In the lungs and liver, the activation of the oxidative enzymes myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and gp91(phox), the production of free radicals, lipid peroxidation, and cell death were substantially and similarly reduced. Treatment effects were less robust in the kidney. Overall, the efficacy of the anti-α4β1 treatment did not differ greatly from that of the anti-CD11d antibody, although details of the results differed. The SIRS after SCI impedes recovery, and attenuation of the SIRS with an anti-integrin treatment is an important, clinically-relevant finding.

FIG. 1

The anti-α4 treatment decreases neutrophils and macrophages in the lung at 24 h after spinal cord injury (SCI). (A) Photomicrographs of lung sections immunostained by an anti-neutrophil antibody (panels 1–3), and by an ED-1 antibody to detect macrophages (panels 4–6) from an uninjured rat, a T4 SCI control rat, and a T4 SCI rat treated with the anti-α4 monoclonal antibody (SCI anti-α4 mAb). The insets in A3 and A6 show high-power detail of stained cells (a, alveolus). The arrows in A2 and A5 point to a neutrophil and a macrophage, respectively (scale bar = 100 μm in A6 applies to A1–A6; scale bar = 10 μm in insets). (B) Neutrophil protein, identified by Western blotting in lung homogenates from uninjured and SCI rats (n = 4 for all groups) expressed in arbitrary units (A.U.; U, uninjured rats; T4C, T4 control SCI rats; T4T, T4 SCI rats treated with the anti-α4 mAb). A representative autoradiogram of a Western blot showing relative protein expression, compared to loading controls (β-actin), is shown above the bar graph. (C) Myeloperoxidase (MPO) activity in lung homogenates from uninjured rats (n = 6), T4 SCI control rats (n = 4), and T4 SCI rats treated with the anti-α4 mAb (n = 5). (D) Macrophage protein (ED-1) expression (Western blotting) in lung homogenates from uninjured and SCI rats (n = 4/group). In this and all figures values are means ± standard error (*significantly different from uninjured; #significantly different from SCI control; p ≤ 0.05 by Student Neuman-Keuls test for all comparisons).

FIG. 2

The anti-α4 treatment decreases expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and gp91^phox, and production of free radicals in the lung at 24 h after spinal cord injury (SCI). (A) iNOS expression was examined in uninjured rats and SCI rats (n = 4 for all groups; U, uninjured rats; T4C, T4 control SCI rats; T4T, T4 SCI rats treated with the anti-α4 monoclonal antibody [mAb]; A.U., arbitrary units). (B and C) COX-2 and gp91^phox expression was also evaluated in these rats (n = 4 for all groups). (D) The concentration of 2′-7′-dichlorofluorescein (DCF) was assayed as a free radical marker in lung homogenates from the uninjured (n = 6), T4C (n = 4), and T4T (n = 5) rats (*significantly different from uninjured; #significantly different from SCI controls, p ≤ 0.05 by Student Neuman-Keuls test).

FIG. 3

The anti-α4 treatment decreases lipid peroxidation and cell death in the lung at 24 h after spinal cord injury (SCI). (A) Lipid peroxidation was assessed by the thiobarbituric acid reactive substance (TBARS) assay for aldehydes, including malondialdehyde in lung homogenates from uninjured (n = 6), T4C (n = 4), and T4T (n = 5) rats, and also by Western blotting for 4-hydroxynonenol (HNE)-bound proteins (B) in most of these rats (n = 4 per group). Western blot illustrates an example of expression of HNE-bound proteins with different molecular weights. The bar graphs display the sums of areas of all bands. (C) Caspase-3 expression was also evaluated by Western blotting in the homogenates from these rats (n = 4 for all groups; *significantly different from uninjured; #significantly different from T4 SCI controls; p ≤ 0.05 by Student Neuman-Keuls test; U, uninjured rats; T4C, T4 control SCI rats; T4T, T4 SCI rats treated with the anti-α4 monoclonal antibody [mAb]; A.U., arbitrary units).

FIG. 4

The anti-α4 treatment decreases neutrophils and macrophages in the kidney at 24 h after spinal cord injury (SCI). (A) Photomicrographs of kidney sections from uninjured and T4 SCI rats, immunostained by the anti-neutrophil antibody (A1–A3), and by the ED-1 antibody (A4–A6). Insets in A2 and A6 show high-power detail of cells with morphology typical of neutrophils and macrophages, respectively (g, glomerulus; t, tubule). Arrows in A2 point to neutrophils in the glomerulus and near a tubule. Arrow in A5 points to a macrophage near a tubule (scale bar = 100 μm in A6 also applies to A1–A6; scale bar = 10 μm in insets). (B) Neutrophil protein expression was examined by Western blotting in uninjured and T4 SCI rats (n = 4 for all groups). (C) Macrophage protein (ED-1) expression was also detected by Western blotting in these rats (n = 4 per group; *significantly different from uninjured; #significantly different from T4 SCI controls; U, uninjured rats; T4C, T4 control SCI rats; T4T, T4 SCI rats treated with the anti-α4 monoclonal antibody [mAb]; A.U., arbitrary units).

FIG. 5

The anti-α4 treatment decreases the production of free radicals and lipid peroxidation in the kidney and liver at 24 h after spinal cord injury (SCI). (A and C) The concentration of 2′-7′-dichlorofluorescein (DCF) was assayed as a free radical marker in the kidney (A) and liver (C) homogenates from the uninjured (n = 6), T4C (n = 4), and T4T (n = 5) rats. (B and D) Lipid peroxidation was assessed by the thiobarbituric acid reactive substance (TBARS) assay for aldehydes, including malondialdehyde, in lung homogenates from the same groups of rats (group numbers the same as those in A and C; *significantly different from uninjured; #significantly different from T4 SCI controls; + tended to differ from T4 controls; p = 0.063; U, uninjured rats; T4C, T4 control SCI rats; T4T, T4 SCI rats treated with the anti-α4 monoclonal antibody [mAb]).

FIG. 6

The anti-α4 treatment decreases neutrophils and macrophages in the liver at 24 h after spinal cord injury (SCI). (A) Photomicrographs of liver sections from uninjured and T4 SCI rats, immunostained by the anti-neutrophil antibody (A1–A3), and by the ED-1 antibody (A4–A6). Insets in A3 and A6 show high-power detail of stained cells (s, liver sinusoid). Arrows point to neutrophils (A2 and A3) and macrophages (A5 and A6; scale bar = 100 μm in A6 also applies to A1–A6; scale bar = 10 μm in insets of A3 and A6). (B) Neutrophil protein expression was revealed by Western blotting in uninjured and T4 SCI rats (n = 4 for all groups). (C) Myeloperoxidase (MPO) activity was assayed in liver homogenates of uninjured (n = 6), T4C rats (n = 4), and T4T rats (n = 5). (D) Macrophage protein (ED-1) expression was also examined by Western blotting in these rats (n = 4 for all groups; *significantly different from uninjured; #significantly different from T4 SCI control; + tended to differ from T4 controls; p = 0.065; U, uninjured rats; T4C, T4 control SCI rats; T4T, T4 SCI rats treated with the anti-α4 monoclonal antibody [mAb]; A.U., arbitrary units).