CD11d integrin blockade reduces the systemic inflammatory response syndrome after spinal cord injury

Abstract

Traumatic injury to the spinal cord triggers a systemic inflammatory response syndrome (SIRS), in which inflammatory cells from the circulation invade organs such as the liver, lung and kidney, leading to damage of these organs. Our previous study (Gris, et al, Exp. Neurol, 2008) demonstrated that spinal cord injury (SCI) activates circulating neutrophils that then invade the lung and kidney from 2 to 24 h after injury, increasing myeloperoxidase activity, cyclooxygenase-2 and matrix metalloproteinase-9 expression and lipid peroxidation in these organs. The present study was designed to ascertain whether a treatment that limits the influx of leukocytes into the injured spinal cord would also be effective in reducing the SIRS after SCI. This treatment is intravenous delivery of a monoclonal antibody (mAb) against the CD11d subunit of the CD11d/CD18 integrin expressed by neutrophils and monocytes. We delivered the anti-CD11d mAb at 2 h post moderate clip compression SCI at the 4th or 12th thoracic segments and assessed inflammation, oxidative activity and cellular damage within the lung, kidney and liver at 12 h post-injury. In some analyses we compared high and low thoracic injuries to evaluate the importance of injury level on the intensity of the SIRS. After T4 injury, treatment with the anti-integrin mAb reduced the presence of neutrophils and macrophages in the lung, with associated decreases in expression of NF-κB and oxidative enzymes and in the concentration of free radicals in this organ. The treatment also reduced lipid peroxidation, protein nitration and cell death in the lung. The anti-CD11d treatment also reduced the inflammatory cells within the kidney after T4 injury, as well as the free radical concentration and amount of lipid peroxidation. In the liver, the mAb treatment reduced the influx of neutrophils but most of the other measures examined were unaffected by SCI. The inflammatory responses within the lung and kidney were often greater after T4 than T12 injury. Clinical studies show that SIRS, with its associated organ failure, contributes significantly to the morbidity and mortality of SCI patients. This anti-integrin treatment may block the onset of SIRS after SCI.

Fig. 1

The anti-CD11d treatment decreases neutrophils and macrophages in the lung at 12 h after SCI. A) Photomicrograph 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-CD11d mAb (SCI anti-CD11d). Insets in A2 and A5 show high power detail of stained cells. a, alveolus; bv, blood vessel. Scale bar=100 μm in A3 (applies to A1–3) and A6 (applies to A4–6) and 10 μm in insets. B) MPO activity in lung homogenates from T4 and T12 SCI rats (n=5 per group) and from uninjured rats (n=6). C) Neutrophil protein, identified by Western blotting in lung homogenates from uninjured and SCI rats (n=5, all groups) expressed in arbitrary units (A.U.). A representative autoradiogram of a Western blot showing relative protein expression, compared to loading controls (β-actin), is shown above the bar graph. D) Macrophage protein (ED-1) expression (Western blotting) in lung homogenates from uninjured and SCI rats (n=4, SCI controls; n=5, uninjured and anti-CD11d treated). In this and all figures: values are means±S.E.; symbols indicating significance refer to the ‘a posteriori’ Student Neuman Keul’s test for all comparisons (P≤0.05) *, significantly different from uninjured; #, significantly different from SCI control; ●, significantly different from T12 SCI control. , tended to differ from T12 SCI anti-CD11d treated. U: uninjured; T4C: T4 SCI control; T4T: T4 SCI anti-CD11d-treated; T12C: T12 SCI control; T12T: T12 SCI anti-CD11d-treated.

Fig. 2

The anti-CD11d treatment decreases expression of NF-κB, iNOS and COX-2 in the lung at 12 h after SCI. A) Photomicrograph of lung sections immunostained for NF-κB from uninjured and T4 SCI rats (panels A1–3). Inset in A2 shows detail of a cell resembling a macrophage with a stained nucleus. Many cells also resembled neutrophils. Arrow indicates clusters of cells next to alveolus. Scale bar=100 μm in A3 (applies to A1–3) and 10 μm in inset of A2. B) NF-κB was identified by Western blotting in uninjured and T4 SCI rat lungs (n=5, all groups). C) iNOS expression was examined in uninjured and T4 or T12 SCI rat lungs (n=4 SCI controls; n=5, uninjured and anti-CD11d treated). D) COX-2 expression was also evaluated in these rats (n=5, all groups). *, significantly different from uninjured; #, significantly different from SCI controls.

Fig. 3

The anti-CD11d treatment decreases expression of gp91^phox and production of free radicals in the lung at 12 h after SCI. A) Photomicrograph of lung sections immunostained for gp91^phox from uninjured and T4 SCI rats (panels A1–3). Inset in A2 shows detail of stained cell resembling a neutrophil (irregular pale nucleus). Scale bar=100 μm in A3 (applies to A1–3) and 10 μm in inset of A2. B) gp91^phox was identified by Western blotting in uninjured rats and in T4 and T12 SCI rat lungs (n=5, all groups). C) The concentration of DCF was assayed as a free radical marker in lung homogenates from uninjured (n=6) and T4 and T12 SCI rats (n=5 per group). *, significantly different from uninjured; #, significantly different from T4 SCI control; #, tendency for significant difference from T4 SCI control.

Fig. 4

The anti-CD11d treatment decreases lipid peroxidation in the lung at 12 h after SCI. A) Lipid peroxidation was assessed by the TBARS assay for malondialdehyde (MDA) in lung homogenates from uninjured (n=6) and T4 or T12 SCI rats (n=5 per group). B) Lipid peroxidation was also assayed by Western blotting for 4-hydroxynonenol (HNE)-bound proteins in these groups of rat lungs (n=5 per group). Western blot illustrates an example of HNE-bound protein expression at several molecular weights. *, significantly different from uninjured; #, significantly different from T4 SCI control; , T4 SCI control tended to differ from T12 SCI control.

Fig. 5

The anti-CD11d treatment decreases protein nitration and caspase-3 expression in the lung at 12 h after SCI. A) Photomicrograph of lung sections immunostained for nitrotyrosine (Ntyr) from uninjured and T4 SCI rats (panels 1–3). Scale=100 μm in A3. B) Nitrotyrosine was assayed in lung homogenates in uninjured and T4 SCI rats (n=5, all groups). C) Caspase-3 expression was evaluated by Western blotting in uninjured and T4 or T12 SCI rat lungs (n=4 SCI controls; n=5, uninjured and anti-CD11d treated). *, significantly different from uninjured; #, significantly different from T4 SCI control.

Fig. 6

The anti-CD11d treatment decreases neutrophils and macrophages in the kidney at 12 h after SCI. A) Photomicrograph of kidney sections from uninjured and T4 SCI rats, immunostained by the anti-neutrophil antibody (A1–3) and by the ED-1 antibody (A4–6). Insets in A2 and A5 show high power detail of cells with morphology typical of neutrophils and macrophages, respectively. g, glomerulus; t, tubule. Arrow in A1 points to a tubule in cross section. Arrows on remaining photo micrographs explained in text. Scale bar=100 μm in A3 (applies to A1–3) and A6 (applies to A4–6) and 10 μm in insets of A2 and A5. B) MPO activity was assayed in kidney homogenates from uninjured (n=6) and T4 and T12 SCI rats (n=5 both groups). C) Neutrophil protein expression was examined by Western blotting in uninjured and T4 SCI rats (n=5 both groups). D) Macrophage protein (ED-1) expression was also detected by Western blotting in these rat kidneys (n=5 per group). *, significantly different from uninjured; #, significantly different from T4 SCI controls; ●, significant difference between T4 and T12 control SCI rats.

Fig. 7

The anti-CD11d treatment decreases neutrophils but not macrophages in the liver at 12 h after SCI. A) Photomicrograph of liver sections from uninjured and T4 SCI rats, immunostained by the anti-neutrophil antibody (A1–3) and by the ED-1 antibody (A4–6). Insets in A2 and A4 show high power detail of stained cells. s, liver sinusoid. Arrows explained in text. Scale bar=100 μm in A3 (applies to A1–3) and A6 (applies to A4–6), and 10 μm in insets of A2 and 5. B) MPO activity was assayed in liver homogenates of uninjured (n=6) and SCI rats with injury at T4 or T12 (n=5 all groups). C) Neutrophil protein expression was revealed by Western blotting in uninjured and T4 SCI rat livers (n=5 both groups). D) Macrophage protein (ED-1) expression was also examined by Western blotting in these rat livers (n=5 both groups). *, significantly different from uninjured; #, significantly different from T4 SCI control.