CD11d integrin blockade reduces the systemic inflammatory response syndrome after traumatic brain injury in rats

Abstract

Traumatic CNS injury triggers a systemic inflammatory response syndrome (SIRS), in which circulating inflammatory cells invade body organs causing local inflammation and tissue damage. We have shown that the SIRS caused by spinal cord injury is greatly reduced by acute intravenous treatment with an antibody against the CD11d subunit of the CD11d/CD18 integrin expressed by neutrophils and monocyte/macrophages, a treatment that reduces their efflux from the circulation. Traumatic brain injury (TBI) is a frequently occurring injury after motor vehicle accidents, sporting and military injuries, and falls. Our studies have shown that the anti-CD11d treatment diminishes brain inflammation and oxidative injury after moderate or mild TBI, improving neurological outcomes. Accordingly, we examined the impact of this treatment on the SIRS triggered by TBI. The anti-CD11d treatment was given at 2h after a single moderate (2.5-3.0 atm) or 2 and 24h after each of three consecutive mild (1.0-1.5 atm) fluid percussion TBIs. Sham-injured, saline-treated rats served as controls. At 24h, 72 h, and 4 or 8 weeks after the single TBI and after the third of three TBIs, lungs of rats were examined histochemically, immunocytochemically and biochemically for downstream effects of SIRS including inflammation, tissue damage and expression of oxidative enzymes. Lung sections revealed that both the single moderate and repeated mild TBI caused alveolar disruption, thickening of inter-alveolar tissue, hemorrhage into the parenchyma and increased density of intra-and peri-alveolar macrophages. The anti-CD11d treatment decreased the intrapulmonary influx of neutrophils and the density of activated macrophages and the activity of myeloperoxidase after these TBIs. Moreover, Western blotting studies showed that the treatment decreased lung protein levels of oxidative enzymes gp91(phox), inducible nitric oxide synthase and cyclooxygenase-2, as well as the apoptotic pathway enzyme caspase-3 and levels of 4-hydroxynonenal-bound proteins (an indicator of lipid peroxidation). Decreased expression of the cytoprotective transcription factor Nrf2 reflected decreased lung oxidative stress. Anti-CD11d treatment also diminished the lung concentration of free radicals and tissue aldehydes. In conclusion, the substantial lung component of the SIRS after single or repeated TBIs is significantly decreased by a simple, minimally invasive and short-lasting anti-inflammatory treatment.

Keywords: Anti-integrin treatment; Lung; Lung damage; Systemic inflammatory response; Traumatic brain injury.

Fig. 1

Moderate or mild TBI leads to disruption of normal lung structure. Photomicrographs (60×) illustrate 25 μm thick sections of hematoxylin and eosin-stained lung sampled 24 h after a sham or a single moderate TBI (A, B) and 72 h after the third of three sham or mild TBIs (C, D). A) Normal alveoli (a) and alveolar sacs (sac) are shown bordered by alveolar septa (as) and inter-alveolar epithelial and connective tissue. Alveolar and peri-alveolar epithelial cells have dark round nuclei and pink cytoplasm. Peri-alveolar macrophages (examples indicated by white arrowheads) are large cells with blue cytoplasm and dark blue nuclei. B) At 24 h after a single moderate TBI, the peri-alveolar tissue is disrupted, containing many erythrocytes (examples indicated by white rbc with arrows) demonstrating intrapulmonary hemorrhage, and a large number of macrophages. C) Normal lung structure at 72 h after the third sham TBI. D) At 72 h after the third mild TBI, the peri-alveolar tissue is compacted, alveolar walls appear disrupted, alveolar septa are thickened, small areas of hemorrhage are present and the density of peri- and intra-alveolar macrophages is greater than in the examples from the lung after sham injury. Scale bars = 20 μm.

Fig. 2

The anti-CD11d treatment decreases neutrophils in the lung at 24 and 72 h after a single moderate TBI and 72 h after the third of three mild repeated TBIs. A) MPO activity in lung homogenates from sham-injured (sham), 1B7-antibody treated control TBI rats (1B7) and anti-CD11d-treated TBI rats (CD11d). Numbers of rats per group are in Table 1. B) Neutrophil protein, identified by Western blotting in lung homogenates from sham-injured and TBI rats, expressed as a ratio of the optical density/mm² of the 56 kDa neutrophil protein band to the optical density/mm² of the corresponding β-actin band. A representative autoradiogram of a Western blot of the 56 kDa protein is shown below the bar graphs. In this and all other figures: values are means ± S.E.; *significantly different from sham-injured; ^#significantly different from IB7 control; P ≤ 0.05 after Student Neuman Keul’s test for all comparisons. C) Photomicrographs (20×) of lung sections immunostained by an anti-neutrophil antibody from sham-injured rats (left panels), IB7 control TBI rats (middle panels) and TBI rats treated with the anti-CD11d mAb (right panels). The top row illustrates examples at 24 h after a single moderate TBI and the bottom row shows examples at 72 h after the third of three mild TBIs. Upper sham injury panel shows a high power (100×) detail of a stained cell (inset). An alveolus (a) and a blood vessel (bv) are indicated in the top sham injury panel. Scale bar is 100 μm and applies to all photomicrographs. Scale bar in the inset is 10 μm.

Fig. 3

The anti-CD11d treatment decreases macrophages in the lung at 24 and 72 h after a single moderate TBI and 72 h after the third of three mild repeated TBIs. A) Macrophage protein (ED-1) expression (Western blotting) in lung homogenates from sham-injured rats (sham, n = 5), 1B7-antibody treated control TBI rats (1B7, n = 5) and anti-CD11d-treated TBI rats (CD11d, n = 5). Figure format is as in Fig. 2B. *Significantly different from sham-injured; ^#significantly different from 1B7 controls. B) Photomicrographs (20×) of lung sections immunostained by an anti-ED-1 antibody to detect macrophages in lungs from sham-injured rats (left panels), IB7 control TBI rats (middle panels) and TBI rats treated with the anti-CD11d mAb (right panels). The top row illustrates examples at 24 h after a single moderate TBI and the bottom row shows examples at 72 h after the third of three mild TBIs. The inset in the upper sham panel shows a high power (100×) detail of a stained cell. In the upper CD11d panel an alveolus (a) and a blood vessel (bv) are indicated. Scale bar in the upper sham panel is 100 μm and applies to all photomicrographs. Scale bar in the inset is 10 μm. Numbers of rats per group studied by immunostaining are in Table 1.

Fig. 4

Treatment with the monoclonal antibody causes no direct inflammatory response within the lung. Comparisons are made between MPO activity in the lungs of untreated, 1B7 control mAb-treated and saline-treated rats that were uninjured, sham-injured or injured by a single moderate TBI. Numbers of rats per group are in Table 1. The saline treated sham-injured group (n = 6) is from Fig. 2. Groups designated with an ‘a’ are not different from each other, groups with a ‘b’ are not different from each other but all groups ‘a’ differ from both groups ‘b’ (P < 0.0001).

Fig. 5

The anti-CD11d treatment decreases expression of gp91^phox and iNOS in the lung at 24 and 72 h after a single moderate TBI and 72 h after the third of three mild repeated TBIs. gp91^phox (A) and iNOS (B) expression were identified by Western blotting in lung homogenates from sham-injured, 1B7-treated TBI and anti-CD11d treated TBI rats (n = 5 per group) and are expressed as ratios of the optical density/mm² of the gp91^phox and iNOS bands to the optical density/mm² of the corresponding β-actin bands. Representative autoradiograms of Western blots of the gp91^phox and iNOS proteins are shown below the bar graphs. Figure format is as in Fig. 2B. *Significantly different from sham-injured; ^#significantly different from 1B7 controls.

Fig. 6

The anti-CD11d treatment decreases lipid peroxidation in the lung at 24 and 72 h after a single moderate TBI and 72 h after the third of three mild repeated TBI. A) Lipid peroxidation was assessed by the TBARS assay for malondialdehyde (MDA) and other aldehydes in lung homogenates from sham-injured, 1B7-treated TBI and anti-CD11d treated TBI rats. Numbers of rats per group are in Table 1. Figure format is as in Fig. 2A. B) Lipid peroxidation was also assayed by Western blotting for 4-hydroxynonenol (HNE)-bound proteins in these groups of rats (n = 5 per group). Format is as in Fig. 2B. Western blot illustrates an example of HNE-bound protein expression at several molecular weights. *Significantly different from sham-injured; ^#significantly different from 1B7 controls.

Fig. 7

The anti-CD11d treatment decreases expression of the cytoprotective transcription factor Nrf2 and of caspase-3 expression in the lung at 24 and 72 h after a single moderate TBI and 72 h after the third of three mild repeated TBI. Nrf2 (A) and caspase-3 (B) expression were identified by Western blotting in lung homogenates from sham-injured, 1B7-treated TBI and anti-CD11d treated TBI rats (n = 5 per group). Numbers of rats studied by immunostaining are in Table 1. Figure format is as in Fig. 2B. *Significantly different from uninjured; ^#significantly different from T4 SCI control.