ß2 Integrin CD11d/CD18: From Expression to an Emerging Role in Staged Leukocyte Migration.

CD11d/CD18 is the most recently discovered and least understood ß2 integrin. Known CD11d adhesive mechanisms contribute to both extravasation and mesenchymal migration - two key aspects for localizing peripheral leukocytes to sites of inflammation. Differential expression of CD11d induces differences in monocyte/macrophage mesenchymal migration including impacts on macrophage sub-set migration. The participation of CD11d/CD18 in leukocyte localization during atherosclerosis and following neurotrauma has sparked interest in the development of CD11d-targeted therapeutic agents. Whereas the adhesive properties of CD11d have undergone investigation, the signalling pathways induced by ligand binding remain largely undefined. Underlining each adhesive and signalling function, CD11d is under unique transcriptional control and expressed on a sub-set of predominately tissue-differentiated innate leukocytes. The following review is the first to capture the nearly three decades of CD11d research and discusses the emerging role of CD11d in leukocyte migration and retention during the progression of a staged immune response.

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

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.

Temporal changes in monocyte and macrophage subsets and microglial macrophages following spinal cord injury in the Lys-Egfp-ki mouse model.

The role of hematogenous (hMΦ) and microglial (mMΦ) macrophages following spinal cord injury (SCI) remains unclear as they are not distinguished easily from each other in the lesion area. We have recently described the temporal and spatial response to SCI of each MΦ population using the lys-EGFP-ki mouse that enables EGFP(+) hMΦ to be distinguished from EGFP(-) mMΦ at the lesion site. In the present study, we characterized the response of monocyte and hMΦ subsets and mMΦ to SCI. We describe, for the first time, the responses of circulating classical (pro-inflammatory) and non-classical monocyte subsets to SCI. Additionally, we show the presence of classical and non-classical hMΦ at the SCI lesion. Importantly, we demonstrate that the 'classical pro-inflammatory' hMΦ respond in the acute (1d, 3d) stages of SCI while the 'non-classical' hMΦ respond in the sub-acute (7d, 14d) phase of SCI. At later time points (6weeks post injury) classical hMΦ return to the injury site. Our study offers new insight into the cellular inflammatory response that occurs after SCI and suggests that the timing and targets of anti-inflammatory therapies may be crucial to maximize neuroprotection at the acute and more chronic stages of SCI.

Treatment with an anti-CD11d integrin antibody reduces neuroinflammation and improves outcome in a rat model of repeated concussion.

BACKGROUND: Concussions account for the majority of traumatic brain injuries (TBI) and can result in cumulative damage, neurodegeneration, and chronic neurological abnormalities. The underlying mechanisms of these detrimental effects remain poorly understood and there are presently no specific treatments for concussions. Neuroinflammation is a major contributor to secondary damage following more severe TBI, and recent findings from our laboratory suggest it may be involved in the cumulative properties of repeated concussion. We previously found that an anti-CD11d monoclonal antibody that blocks the CD11d/CD18 integrin and adhesion molecule interaction following severe experimental TBI reduces neuroinflammation, oxidative activity, and tissue damage, and improves functional recovery. As similar processes may be involved in repeated concussion, here we studied the effects of the anti-CD11d treatment in a rat model of repeated concussion. METHODS: Rats were treated 2 h and 24 h after each of three repeated mild lateral fluid percussion injuries with either the CD11d antibody or an isotype-matched control antibody, 1B7. Injuries were separated by a five-day inter-injury interval. After the final treatment and either an acute (24 to 72 h post-injury) or chronic (8 weeks post-injury) recovery period had elapsed, behavioral and pathological outcomes were examined. RESULTS: The anti-CD11d treatment reduced neutrophil and macrophage levels in the injured brain with concomitant reductions in lipid peroxidation, astrocyte activation, amyloid precursor protein accumulation, and neuronal loss. The anti-CD11d treatment also improved outcome on tasks of cognition, sensorimotor ability, and anxiety. CONCLUSIONS: These findings demonstrate that reducing inflammation after repeated mild brain injury in rats leads to improved behavioral outcomes and that the anti-CD11d treatment may be a viable therapy to improve post-concussion outcomes.

Disordered cardiovascular control after spinal cord injury.

Damage to the spinal cord disrupts autonomic pathways, perturbing cardiovascular homeostasis. Cardiovascular dysfunction increases with higher levels of injury and greater severity. Disordered blood pressure control after spinal cord injury (SCI) has significant ramifications as cord-injured people have an increased risk of developing heart disease and stroke; cardiovascular dysfunction is currently a leading cause of death among those with SCI. Despite the clinical significance of abnormal cardiovascular control following SCI, this problem has been generally neglected by both the clinical and research community. Both autonomic dysreflexia and orthostatic hypotension are known to prevent and delay rehabilitation, and significantly impair the overall quality of life after SCI. Starting with neurogenic shock immediately after a higher SCI, ensuing cardiovascular dysfunctions include orthostatic hypotension, autonomic dysreflexia and cardiac arrhythmias. Disordered temperature regulation accompanies these autonomic dysfunctions. This chapter reviews the human and animal studies that have furthered our understanding of the pathophysiology and mechanisms of orthostatic hypotension, autonomic dysreflexia and cardiac arrhythmias. The cardiovascular dysfunction that occurs during sexual function and exercise is elaborated. New awareness of cardiovascular dysfunction after SCI has led to progress toward inclusion of this important autonomic problem in the overall assessment of the neurological condition of cord-injured people.

A CD11d monoclonal antibody treatment reduces tissue injury and improves neurological outcome after fluid percussion brain injury in rats.

Traumatic brain injury (TBI) is an international health concern often resulting in chronic neurological abnormalities, including cognitive deficits, emotional disturbances, and motor impairments. An anti-CD11d monoclonal antibody that blocks the CD11d/CD18 integrin and vascular cell adhesion molecule (VCAM)-1 interaction following experimental spinal cord injury improves functional recovery, while reducing the intraspinal number of neutrophils and macrophages, oxidative activity, and tissue damage. Since the mechanisms of secondary injury in the brain and spinal cord are similar, we designed a study to evaluate fully the effects of anti-CD11d treatment after a moderate lateral fluid percussion TBI in the rat. Rats were treated at 2 h after TBI with either the anti-CD11d antibody or an isotype-matched control antibody 1B7, and both short (24- to 72-h) and long (4-week) recovery periods were examined. The anti-CD11d integrin treatment reduced neutrophil and macrophage levels in the injured brain, with concomitant reductions in lipid peroxidation, astrocyte activation, amyloid precursor protein accumulation, and neuronal loss. The reduced neuroinflammation seen in anti-CD11d-treated rats correlated with improved performance on a number of behavioral tests. At 24 h, the anti-CD11d group performed significantly better than the 1B7 controls on several water maze measures of spatial cognition. At 4 weeks post-injury the anti-CD11d-treated rats had better sensorimotor function as assessed by the beam task, and reduced anxiety-like behaviors, as evidenced by elevated-plus maze testing, compared to 1B7 controls. These findings suggest that neuroinflammation is associated with behavioral deficits after TBI, and that anti-CD11d antibody treatment is a viable strategy to improve neurological outcomes after TBI.

Differential detection and distribution of microglial and hematogenous macrophage populations in the injured spinal cord of lys-EGFP-ki transgenic mice.

The acute inflammatory response that follows spinal cord injury (SCI) contributes to secondary injury that results in the expansion of the lesion and further loss of neurologic function. A cascade of receptor-mediated signaling events after SCI leads to activation of innate immune responses including the migration of microglia and active recruitment of circulating leukocytes. Because conventional techniques do not always distinguish macrophages derived from CNS-resident microglia from blood-derived monocytes, the role that each macrophage type performs cannot be assessed unambiguously in these processes. We demonstrate that, in the normal and spinal cord-injured lys-EGFP-ki transgenic mouse, enhanced green fluorescent protein (EGFP) is expressed only in mature hematopoietic granulomyelomonocytic cells and not in microglia. This allowed us to assess the temporal and spatial relationships between microglia-derived and hematogenous macrophages as well as neutrophils during a period of 6 weeks after clip compression SCI. Within the lesion, EGFP-positive monocyte-derived macrophages were found at the epicenter surrounded by EGFP-negative-activated microglia and microglia-derived macrophages. Neutrophils were not present when EGFP-positive monocyte-derived macrophages were depleted, indicating that neutrophil persistence in the lesion depended on the presence of these monocytes. Thus, these 2 distinct macrophage populations can be independently identified and tracked, thereby allowing their roles in acute and chronic stages of SCI-associated inflammation to be defined.

The dark side of neuroplasticity.

Whether dramatic or modest, recovery of neurological function after spinal cord injury (SCI) is greatly due to neuroplasticity--the process by which the nervous system responds to injury by establishing new synaptic connections or by altering the strength of existing synapses. However, the same neuroplasticity that allows locomotor function to recover also produces negative consequences such as pain and dysfunction of organs controlled by the autonomic nervous system. In this review we focus specifically on structural neuroplasticity (the growth of new synaptic connections) after SCI and on the consequent development of pain and autonomic dysreflexia, a condition of episodic hypertension. Neuroplasticity after SCI is stimulated by the deafferentation of spinal neurons below the lesion and by the expression of growth-promoting neurotrophins such as nerve growth factor (NGF). A broad range of therapeutic strategies that affect neuroplasticity is being developed for the treatment of SCI. At one end of the spectrum are therapeutic strategies that directly or indirectly increase NGF in the injured spinal cord, and have the most robust effects on neuroplasticity. At the other end of the spectrum are neuroprotective strategies focused on supporting and rescuing uninjured, or partially injured, axons; these might limit the deafferentation stimulus for neuroplasticity. In the middle of this spectrum are strategies that block axon growth inhibitors without necessarily providing a growth stimulus. The literature supports the view that the negative consequences of neuroplasticity develop more commonly with therapies that directly stimulate nerve growth than they develop in the untreated injured cord. Compared to these conditions, neuroplasticity with negative outcomes is less prevalent after treatments that that neutralize axon growth inhibitors, and least apparent after strategies that promote neuroprotection.

Anti-CD11d monoclonal antibody treatment for rat spinal cord compression injury.

This paper by Hurtado et al. examined responses of spinal cord-injured rats to treatment with a monoclonal antibody to the CD11d integrin, as a replication study of the paper by Gris et al. published in J. Neuroscience, 2004. The Hurtado et al. study addressed a portion of our investigation and obtained similar findings in the experiments that closely replicated ours in methodology and design, specifically the open field locomotor study. The high variability in their study of mechanical allodynia probably precluded detection of effects of the anti-CD11d treatment on this form of neuropathic pain. The lesion assessments were greatly different from those done in the Gris et al. study, and may not have been ideal for the extent of injury produced in this model, but did reveal a trend toward myelin preservation. The positive aspects of the study by Hurtado et al. encourage us to investigate this novel treatment further, in different animals and in different models of spinal cord injury.

The systemic inflammatory response after spinal cord injury in the rat is decreased by α4ß1 integrin blockade.

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 α4ß1 integrin limited secondary cord damage more effectively than blockade of CD11d/CD18. Therefore we considered it important to assess the effects of anti-α4ß1 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-α4ß1 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.

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

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.

A selective phosphodiesterase-4 inhibitor reduces leukocyte infiltration, oxidative processes, and tissue damage after spinal cord injury.

We tested the hypothesis that a selective phosphodiesterase type 4 inhibitor (PDE4-I; IC486051) would attenuate early inflammatory and oxidative processes following spinal cord injury (SCI) when delivered during the first 3 days after injury. Rats receiving a moderately severe thoracic-clip-compression SCI were treated with the PDE4-I (0.5, 1.0, and 3.0 mg/kg IV) in bolus doses from 2-60 h post-injury. Doses at 0.5 mg/kg and 1.0 mg/kg significantly decreased myeloperoxidase (MPO) enzymatic activity (neutrophils), expression of a neutrophil-associated protein and of ED-1 (macrophages), and estimates of lipid peroxidation in cord lesion homogenates at 24 h and 72 h post-injury by 25-40%. The 3.0 mg/kg dose had small or no effects on these measures. The PDE4-I treatment (0.5 or 1.0 mg/kg) reduced expression of the oxidative enzymes gp91(phox), inducible nitric oxide synthase, and cyclooxygenase-2, and diminished free radical generation by up to 40%. Treatment with 0.5 mg/kg PDE4-I improved motor function (as assessed by the Basso-Beattie-Bresnahan scale) significantly from 4-8 weeks after SCI (average difference 1.3 points). Mechanical allodynia elicited from the hindpaw decreased by up to 25%. The PDE4-I treatment also increased white matter volume near the lesion at 8 weeks after SCI. In conclusion, the PDE4-I reduced key markers of oxidative stress and leukocyte infiltration, producing cellular protection, locomotor improvements, and a reduction in neuropathic pain. Early inhibition of PDE4 is neuroprotective after SCI when given acutely and briefly at sufficient doses.

A systematic review of non-invasive pharmacologic neuroprotective treatments for acute spinal cord injury.

An increasing number of therapies for spinal cord injury (SCI) are emerging from the laboratory and seeking translation into human clinical trials. Many of these are administered as soon as possible after injury with the hope of attenuating secondary damage and maximizing the extent of spared neurologic tissue. In this article, we systematically review the available pre-clinical research on such neuroprotective therapies that are administered in a non-invasive manner for acute SCI. Specifically, we review treatments that have a relatively high potential for translation due to the fact that they are already used in human clinical applications, or are available in a form that could be administered to humans. These include: erythropoietin, NSAIDs, anti-CD11d antibodies, minocycline, progesterone, estrogen, magnesium, riluzole, polyethylene glycol, atorvastatin, inosine, and pioglitazone. The literature was systematically reviewed to examine studies in which an in-vivo animal model was utilized to assess the efficacy of the therapy in a traumatic SCI paradigm. Using these criteria, 122 studies were identified and reviewed in detail. Wide variations exist in the animal species, injury models, and experimental designs reported in the pre-clinical literature on the therapies reviewed. The review highlights the extent of investigation that has occurred in these specific therapies, and points out gaps in our knowledge that would be potentially valuable prior to human translation.

A systematic review of directly applied biologic therapies for acute spinal cord injury.

An increasing number of therapies for spinal cord injury (SCI) are emerging from the laboratory and seeking translation into human clinical trials. Many of these are administered as soon as possible after injury with the hope of attenuating secondary damage and maximizing the extent of spared neurologic tissue. In this article, we systematically reviewed the available preclinical research on such neuroprotective therapies that are administered in a non-invasive manner for acute SCI. Specifically, we reviewed treatments that have a relatively high potential for translation due to the fact that they are already used in human clinical applications or are available in a form that could be administered to humans. These included: erythropoietin, NSAIDs, anti-CD11d antibodies, minocycline, progesterone, estrogen, magnesium, riluzole, polyethylene glycol, atorvastatin, inosine, and pioglitazone. The literature was systematically reviewed to examine studies in which an in vivo animal model was utilized to assess the efficacy of the therapy in a traumatic spinal cord injury paradigm. Using these criteria, 122 studies were identified and reviewed in detail. Wide variations exist in the animal species, injury models, and experimental designs reported in the preclinical literature on the therapies reviewed. The review highlights the extent of investigation that has occurred in these specific therapies, and points out gaps in our knowledge that would be potentially valuable prior to human translation.

Human spinal cord injury causes specific increases in surface expression of ß integrins on leukocytes.

Spinal cord injury (SCI) activates circulating leukocytes that migrate into the injured cord and bystander organs using adhesion molecule-mediated mechanisms. These cells cause oxidative damage, resulting in secondary injury to the spinal cord, as well as injury to bystander organs. This study was designed to examine, over a 6-h to 2-week period, changes in adhesion molecule surface expression on human peripheral leukocytes after SCI (9 subjects), using as controls 10 uninjured subjects and 6 general trauma patients (trauma controls, TC). Both the percentage of cells expressing a given adhesion molecule and the average level of its expression was quantified for both circulating neutrophils and monocytes. The percentage of neutrophils and monocytes expressing the selectin CD62L was unchanged in TC and SCI patients after injury compared to uninjured subjects. Concurrently, the amount of surface CD62L on neutrophils was decreased in SCI and TC subjects, and on monocytes after SCI. The percentage of neutrophils expressing α4 decreased in TC, but not in SCI, subjects. Likewise, the percentage of neutrophils and monocytes expressing CD11d decreased markedly in TC subjects, but not after SCI. In contrast, the mean surface expression of α4 and CD11d by neutrophils and monocytes increased after SCI compared with uninjured and TC subjects. The percentage of cells and surface expression of CD11b were similar in neutrophils of all three groups, whereas CD11b surface expression increased after SCI in monocytes. In summary, unlike changes found after general trauma, the proinflammatory stimulation induced by SCI increases the surface expression of adhesion molecules on circulating neutrophils and monocytes before they infiltrate the injured spinal cord and multiple organs of patients. Integrins may be excellent targets for anti-inflammatory treatment after human SCI.

A grading system to evaluate objectively the strength of pre-clinical data of acute neuroprotective therapies for clinical translation in spinal cord injury.

The past three decades have seen an explosion of research interest in spinal cord injury (SCI) and the development of hundreds of potential therapies that have demonstrated some promise in pre-clinical experimental animal models. A growing number of these treatments are seeking to be translated into human clinical trials. Conducting such a clinical trial, however, is extremely costly, not only for the time and money required to execute it, but also for the limited resources that will then no longer be available to evaluate other promising therapies. The decision about what therapies have sufficient pre-clinical evidence of efficacy to justify testing in humans is therefore of utmost importance. Here, we have developed a scoring system for objectively grading the body of pre-clinical literature on neuroprotective treatments for acute SCI. The components of the system include an evaluation of a number of factors that are thought to be important in considering the "robustness" of a therapy's efficacy, including the animal species and injury models that have been used to test it, the time window of efficacy, the types of functional improvements effected by it, and whether efficacy has been independently replicated. The selection of these factors was based on the results of a questionnaire that was performed within the SCI research community. A modified Delphi consensus-building exercise was then conducted with experts in pre-clinical SCI research to refine the criteria and decide upon how to score them. Finally, the grading system was applied to a series of potential neuroprotective treatments for acute SCI. This represents a systematic approach to developing an objective method of evaluating the extent to which the pre-clinical literature supports the translation of a particular experimental treatment into human trials.

Anti-alpha4beta1 integrin antibody induces receptor internalization and does not impair the function of circulating neutrophilic leukocytes.

OBJECTIVE: A compelling strategy for treatment of spinal cord injury is the blockade of integrin-mediated leukocyte extravasation using a monoclonal antibody (mAb) against the alpha4 subunit of the alpha4beta1-integrin. However, little is known with respect to neutrophil function following anti-alpha4 mAb treatment. This study assessed the effects of anti-alpha4 mAb binding on neutrophil activation [reactive oxygen species (ROS) production], function (phagocytic activity) and anti-alpha4-mAb/alpha4beta1-integrin-complex internalization. METHODS: Resting, primed or stimulated rat neutrophils were incubated ex vivo with anti-alpha4 mAb or isotype-control antibody. ROS production, phagocytic activity, and anti-alpha4-mAb/alpha4beta1-integrin-complex internalization were determined by flow cytometry using dihydrorhodamine (DHR1,2,3), fluorescent microspheres, and indirect immunolabeling, respectively. RESULTS: Brief (0.5 h) incubation of resting, primed or activated neutrophils with anti-alpha4 mAb had no effect on ROS production and did not change neutrophil phagocytic activity. However, prolonged incubation (2 h), assessed only in resting neutrophils, increased ROS production. The anti-alpha4-mAb/alpha4beta1-integrin-complex was internalized after 1 h of anti-alpha4 mAb treatment and remained internalized up to 6 h. CONCLUSION: Neutrophil ROS production and phagocytic function remain unaltered after brief anti-alpha4 mAb exposure, demonstrating that use of this mAb as a treatment should not adversely affect important beneficial roles of these cells.

Timing and duration of anti-alpha4beta1 integrin treatment after spinal cord injury: effect on therapeutic efficacy.

OBJECT: After spinal cord injury (SCI) leukocytes infiltrate the injured cord, causing significant damage and further impairment of functional recovery. The leukocyte integrin alpha4beta1 is crucial for their entry. The authors previously demonstrated that an anti-alpha4 monoclonal antibody (mAb) treatment attenuates leukocyte infiltration, improves motor and autonomic function, and reduces neuropathic pain when administered at 2 hours and 24 hours after SCI. METHODS: The authors conducted 2 preclinical studies: the first determined effects of treatment commencing at 6 hours, a clinically relevant time after injury, and the second examined effects of long-lasting treatment (28 days) on neurological recovery after SCI, as current clinically used anti-inflammatory monoclonal antibodies have such longevity. In the first study (timing study), rats were treated with anti-alpha4 or control mAb (intravenously) at 6 hours and 48 hours after moderate (35 g) thoracic compression SCI. Effects on intraspinal inflammation and oxidative injury were assessed at 3 and 7 days after SCI; motor function and pain were examined for 6 weeks. In the second study (duration study), anti-alpha4 mAb was administered starting 2 hours after SCI and subsequently every 3 days for 4 weeks (total of 8 doses), using a schedule of decreasing doses to resemble the pharmacodynamics of long-lasting antibodies used clinically. Motor function and pain were examined for 6 weeks. Lesions were assessed for tissue sparing and inflammation at 6 weeks by histological examination and MR imaging. RESULTS: Anti-alpha4 mAb treatment at 6 hours and 48 hours after SCI (timing study) significantly decreased neutrophil and monocyte/macrophage influx at 3 days by 36% and 20%, respectively, but had no effect by at 7 days after SCI. Antibody treatment significantly reduced intraspinal myeloperoxidase activity by 48% and lipid peroxidation by 27% at 3 days post-injury. The treatment did not improve locomotor function but reduced mechanical allodynia elicited from the trunk and hind paw by ~50% at 3-6 weeks after SCI. In contrast, long-term mAb treatment commencing at 2 hours after SCI (duration study) significantly improved locomotor function at 2-6 weeks after SCI, (mean BBB scores +/- SE: treated rats, 8.3 +/- 0.16; controls, 7.3 +/- 0.2 at 6 weeks). At 3-6 weeks, mAb treatment decreased mechanical allodynia elicited from the trunk and hind paw by ~55%. This recovery correlated with 30% more myelin-containing white matter in treated rats than controls at 6 weeks. The lesion cavity was smaller in the treated rats when assessed by both histological (-37%) and imaging (-50%) methods. The accumulation of ED1-immunoreactive microglia/macrophages at the lesion was similar in treated and control rats. CONCLUSIONS: Although delayed treatment reduced intraspinal inflammation and pain, motor function was not improved, revealing decreased efficacy at the more clinically feasibly treatment onset. Long-term anti-alpha4 mAb treatment starting 2 hours after SCI improved neurological outcomes, with tissue sparing near the lesion and no impairment of the late immune response to injury. These findings reveal no disadvantage of long-lasting immunosuppression by the treatment but show that efficacy depends upon very early delivery.

Increased oxidative activity in human blood neutrophils and monocytes after spinal cord injury.

Traumatic injury can cause a systemic inflammatory response, increasing oxidative activity of circulating leukocytes and potentially exacerbating the original injury, as well as causing damage to initially unaffected organs. Although the importance of intraspinal inflammation after human spinal cord injury is appreciated, the role of the systemic inflammatory response to this injury is not widely recognised. We investigated oxidative activity of blood leukocytes from nine cord-injured subjects and six trauma controls (bone fractures without CNS injury) at 6 h-2 weeks after injury, comparing values to those of ten uninjured subjects. Neutrophil and monocyte free radical production, evaluated by flow cytometry, increased significantly more in cord injury subjects than in trauma controls (6-fold vs 50% increases). In leukocyte homogenates, the concentration of free radicals increased significantly more in cord injury subjects (2-fold) than in the trauma controls (1.6-fold) as did activity of myeloperoxidase (2.3-fold vs. 1.7-fold). Moreover, in homogenates and blood smears, expression of the NADPH oxidase subunit gp91(phox) and of the oxidative enzyme, inducible nitric oxide synthetase was 20-25% greater in cord injury subjects than in trauma controls. Expression of the pro-inflammatory transcription factor NF-kappaB and of cyclooxygenase-2 increased similarly after both injuries. Finally, aldehyde products of tissue-damaging lipid peroxidation also increased significantly more in the plasma of spinal cord injury subjects than in trauma controls (2.6 fold vs. 1.9-fold). Spinal cord injury causes a particularly intense systemic inflammatory response. Limiting this response briefly after cord injury should protect the spinal cord and tissues/organs outside the CNS from secondary damage.

Blockade of the 5-HT3 receptor for days causes sustained relief from mechanical allodynia following spinal cord injury.

Chronic neuropathic pain is a frequent, serious outcome of spinal cord injury (SCI) that is highly refractory to treatment. Serotonin can contribute to neuropathic pain after SCI, as suggested by our previous observation that transient blockade of the 5-HT(3) receptor by intrathecal injections of the antagonist ondansetron reduces mechanical allodynia after SCI in rats. The current study determined whether intrathecal or intravenous infusion of ondansetron for 3 or 7 days, respectively, could cause sustained blockade of mechanical allodynia at and below the level of a twelfth thoracic clip compression injury in rats. Intrathecal 3-day infusion of ondansetron (2.0 microg/hr), targeted to the cord rostral to the SCI and commencing at 28 days after SCI, decreased at-level mechanical allodynia by 40% and below-level allodynia by 60% compared with saline-treated rats (controls). This reduction was sustained throughout drug delivery and for 1 day afterward. During the next 3 days, allodynia gradually returned toward the values of saline-treated rats. An initial experiment showed that bolus intravenous injections of ondansetron (20-100 microg) at 28 days after SCI decreased both at- and below-level allodynia for 90-120 min. Intravenous 7-day infusions (20 microg/hr), commencing at 28 days after SCI, significantly decreased at-level allodynia by 48% and below-level allodynia by 51% compared with controls. This reduction of allodynia lasted throughout the infusion and for 1-3 days afterward while pain responses gradually approached those of controls. These findings suggest a potential role of 5-HT(3) receptor antagonism in the relief of neuropathic pain after SCI in humans.