Pascal Rouleau

Proinflammatory Activities of S100: Proteins S100A8, S100A9, and S100A8/A9 Induce Neutrophil Chemotaxis and Adhesion

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10−12–10−9 M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.

Blockade of S100A8 and S100A9 Suppresses Neutrophil Migration in Response to Lipopolysaccharide

Recently, proinflammatory activities had been described for S100A8 and S100A9, two proteins found at inflammatory sites and within the neutrophil cytoplasm. In this study, we investigated the role of these proteins in neutrophil migration in vivo in response to LPS. LPS was injected into the murine air pouch, which led to the release of S100A8, S100A9, and S100A8/A9 in the pouch exudates that preceded accumulation of neutrophils. Passive immunization against S100A8 and S100A9 led to a 52% inhibition of neutrophil migration in response to LPS at 3 h postinjection. Injection of LPS was also associated with an increase in peripheral blood neutrophils and the presence in serum of S100A9 and S100A8/A9. Intravenous injection of S100A8, S100A9, or S100A8/A9 augmented the number of circulating neutrophils and diminished the number of neutrophils in the bone marrow, demonstrating that S100A8 and S100A9 induced the mobilization of neutrophils from the bone marrow to the blood. Finally, passive immunization with anti-S100A9 inhibited the neutrophilia associated with LPS injection in the air pouch. These results suggest that S100A8 and S100A9 play a role in the inflammatory response to LPS by inducing the release of neutrophils from the bone marrow and directing their migration to the inflammatory site.

Blockade of Antimicrobial Proteins S100A8 and S100A9 Inhibits Phagocyte Migration to the Alveoli in Streptococcal Pneumonia

We investigated the roles of the potent, chemotactic antimicrobial proteins S100A8, S100A9, and S100A8/A9 in leukocyte migration in a model of streptococcal pneumonia. We first observed differential secretion of S100A8, S100A9, and S100A8/A9 that preceded neutrophil recruitment. This is partially explained by the expression of S100A8 and S100A9 proteins by pneumocytes in the early phase of Streptococcus pneumoniae infection. Pretreatment of mice with anti-S100A8 and anti-S100A9 Abs, alone or in combination had no effect on bacterial load or mice survival, but caused neutrophil and macrophage recruitment to the alveoli to diminish by 70 and 80%, respectively, without modifying leukocyte blood count, transendothelial migration or neutrophil sequestration in the lung vasculature. These decreases were also associated with a 68% increase of phagocyte accumulation in lung tissue and increased expression of the chemokines CXCL1, CXCL2, and CCL2 in lung tissues and bronchoalveolar lavages. These results show that S100A8 and S100A9 play an important role in leukocyte migration and strongly suggest their involvement in the transepithelial migration of macrophages and neutrophils. They also indicate the importance of antimicrobial proteins, as opposed to classical chemotactic factors such as chemokines, in regulating innate immune responses in the lung.

The calcium-binding protein S100A12 induces neutrophil adhesion, migration, and release from bone marrow in mouse at concentrations similar to those found in human inflammatory arthritis.

We investigated the proinflammatory activities of S100A12 in the context of synovial inflammation. S100A12 levels were increased in the synovial fluids and plasma of patients with gout, rheumatoid arthritis, psoriatic arthritis, and undetectable in osteoarthritis, a noninflammatory disorder. S100A12 proved to induce neutrophil adhesion to fibrinogen via Mac-1 at concentrations similar to those found in the synovial fluids. Similar concentrations induced the recruitment of large numbers of neutrophils and monocytes in the murine air pouch model. To characterize the effect of increased S100A12 plasma levels, mice were injected intravenously with S100A12. This led to the mobilization of neutrophils from the bone marrow to the peripheral blood. These results suggest that S100A12 stimulates the accumulation of neutrophil by inducing their release from the bone marrow, as well as by activating their adhesion and migration toward inflammatory sites.

Role of spinal 5-HT2 receptor subtypes in quipazine-induced hindlimb movements after a low-thoracic spinal cord transection

A role of serotonin receptors (5‐HTRs) in spinal rhythmogenesis has been proposed several years ago based mainly upon data showing that bath‐applied 5‐HT could elicit locomotor‐like rhythms in in vitro isolated spinal cord preparations. Such a role was partially confirmed in vivo after revealing that systemically administered 5‐HTR2 agonists, such as quipazine, could induce some locomotor‐like movements (LM) in completely spinal cord‐transected (Tx) rodents. However, given the limited binding selectivity of currently available 5‐HTR2 agonists, it has remained difficult to determine clearly if one receptor subtype is specifically associated with LM induction. In situ hybridization, data using tissues from L1–L2 spinal cord segments, where critical locomotor network elements have been identified in mice, revealed greater 5‐HTR2A mRNA levels in low‐thoracic Tx than non‐Tx animals. This expression level remained elevated for several days, specifically in the lateral intermediate zone, where peak values were detected at 1 week post‐Tx and returned to normal at 3 weeks post‐Tx. Behavioral and kinematic analyses revealed quipazine‐induced LM in 1‐week Tx mice either non‐pretreated or pretreated with selective 5‐HTR2B and/or 5‐HTR2C antagonists. In contrast, LM completely failed to be induced by quipazine in animals pretreated with selective 5‐HTR2A antagonists. Altogether, these results provide strong evidence suggesting that 5‐HTR2A are specifically associated with spinal locomotor network activation and LM generation induced by quipazine in Tx animals. These findings may contribute to design drug treatments aimed at promoting locomotor function recovery in chronic spinal cord‐injured patients.

Specific role of dopamine D1 receptors in spinal network activation and rhythmic movement induction in vertebrates

Dopamine (DA) is well‐recognized for its determinant role in the modulation of various brain functions. DA was also found in in vitro isolated invertebrate preparations to activate per se the central pattern generator for locomotion. However, it is less clear whether such a role as an activator of central neural circuitries exists in vertebrate species. Here, we studied in vivo the effects induced by selective DA receptor agonists and antagonists on hindlimb movement generation in mice completely spinal cord‐transected (Tx) at the low‐thoracic level (Th9/10). Administration of D1/D5 receptor agonists (0.5–2.5 mg kg−1, i.p.) was found to acutely elicit rhythmic locomotor‐like movements (LMs) and non‐locomotor movements (NLMs) in untrained and non‐sensory stimulated animals. Comparable effects were found in mice lacking the D5 receptor (D5KO) whereas D1/D5 receptor antagonist‐pretreated animals (wild‐type or D5KO) failed to display D1/D5 agonist‐induced LMs. In contrast, administration of broad spectrum or selective D2, D3 or D4 agonists consistently failed to elicit significant hindlimb movements. Overall, the results clearly show in mice the existence of a role for D1 receptors in spinal network activation and corresponding rhythmic movement generation.

Oral administration of a tri-therapy for central pattern generator activation in paraplegic mice: Proof-of-concept of efficacy

Spinal cord injury (SCI) is a neurological condition, for which no cure exists, typically leading to an immediate and irreversible loss of sensory and voluntary motor functions accompanied by significant health problems. We conducted proof‐of‐concept experiments aimed at assessing efficacy upon oral administration of a novel combination therapy for central pattern generator (CPG) activation and corresponding locomotor movement generation in completely paraplegic animals. Co‐administration orally (by gavage) of buspirone, levodopa and carbidopa was found to dose‐dependently induce episodes of steady weight‐bearing stepping in low‐thoracic (Th9/10) spinal cord‐transected (Tx) mice (with no other form of assistance or training). Robust hindlimb stepping with weight‐bearing capabilities was induced with the tri‐therapy but not with clinically relevant doses of these compounds administered separately. These results provide evidence suggesting that this drug combination may be ideally suited to constitute a first‐in‐class therapy (CPG activator) for locomotor activity induction in chronic SCI individuals, given that efficacy was shown using commercially available brain‐permeable small molecules, already known as safe for the treatment of various neurological indications.

Effects on Locomotion, Muscle, Bone, and Blood Induced by a Combination Therapy Eliciting Weight-Bearing Stepping in Nonassisted Spinal Cord–Transected Mice

Background. The health benefits associated with physical activity–based rehabilitation in patients with no lower-extremity motor function after a spinal cord injury (SCI) is uncertain. Methods. The authors assessed signs of efficacy, safety, and utility associated with a novel pharmacological combination therapy to activate central pattern generator (CPG) activity and corresponding locomotor activity in complete thoracic Th9/10-transected mice. Results. Subcutaneous administration 4 times per week for 1 month of 1.5 mg/kg buspirone, 1.5 mg/kg apomorphine, 12.5 mg/kg benserazide, and 50 mg/kg L-DOPA induced episodes of weight-bearing stepping on a treadmill in nonassisted paraplegic mice for 45-minute sessions. Hindlimb muscle cross-sectional area and fiber area values as well as several blood cell constituent levels assessed at 30 days postinjury were positively affected by the combination therapy, as compared with controls. Episodes of locomotion remained effective on each treatment. Femoral bone mineral density loss was not prevented by triple therapy. Conclusion. Although translation of these findings needs further experimentation, similar pharmacological activation of the CPG offers a novel therapeutic target to provide some health benefits in motor-complete SCI patients.

Traumatic and Non-Traumatic Spinal Cord-Injured Patients in Quebec, Canada: 1. Epidemiological, Clinical and Functional Characteristics

Objective: Differences between traumatic (TSCI) and non-traumatic (NTSCI) spinal cord-injured patients with comparable geographical and socio-economical background have rarely been studied. The objective was to examine and compare a set of epidemiological, clinical, and functional characteristics in TSCI and NTSCI patients. Methods: This is a community-based, cross-sectional study of medical records from a cohort of one hundred and seventy- five (175) chronic spinal cord-injured patients (94 TSCI and 81 NTSCI individuals) who were treated at the Interval Rehabilitation Center located in Trois-Rivieres, Province of Quebec, Canada. Results: Clear differences in age, gender, extent and level of injury or associated conditions (also called secondary complications) were found between TSCI and NTSCI patients. Only one (1/81) completely injured patients was identified among the NTSCI group whereas completely injured patients constituted 37.8% of all TSCI patients. The percentage of patients with associated conditions including neurogenic bladder, neurogenic bowel, urinary tract infection, and pressure ulcer problems was significantly greater in TSCI than NTSCI patients. In contrast, a comparable proportion of TSCI and NTSCI patients was experiencing neuropathic pain. Conclusions: Given these differences between groups as well as regional-specific differences reported in studies from some other countries, it may be suggested that therapeutic approaches developed to treat these health problems and so- called associated conditions could be targeted for specific groups and subgroups of spinal cord-injured patients.

Effects of Spinal α2-Adrenoceptor and I1-Imidazoline Receptor Activation on Hindlimb Movement Induction in Spinal Cord-Injured Mice

A partial recovery of locomotor functions has been shown in spinal cord-transected (Tx) cats after regular treadmill training and repeated administration of clonidine, an α2-adrenoreceptor agonist. However, clonidine has generally failed to show prolocomotor effects in other models (e.g., rat or mudpuppy in vitro-isolated spinal cord preparations). The reasons for this discrepancy remain unclear, but they may suggest condition- or species-specific effects induced by clonidine. This study is aimed at examining both the acute (at 6 or 41 days post-Tx) and chronic effects of repeated (once a week for one month) clonidine administration (0.25–5.0 mg/kg i.p.) on hindlimb movement generation in Tx mice (thoracic segment9/10). Locomotor-like (LM) and nonlocomotor movements (NLM) were assessed both in open-field and treadmill conditions. The results show that clonidine consistently failed, in both conditions, to induce LM and NLM at all time points even though control experiments revealed hindlimb movements steadily induced by 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT), a serotonin receptor agonist. In turn, clonidine acutely suppressed (I1-imidazoline receptor-mediated) the frequency of spontaneously occurring LM and NLM but apparently increased spinal excitability over time, because the frequency of spontaneous LM and NLM was significantly greater in clonidine-treated (before an injection) than vehicle-treated animals after repeated administration for a few weeks. The results clearly show that clonidine can not acutely induce hindlimb movements in untrained and otherwise nonstimulated (e.g., no tail or perineal pinching) Tx mice, although repeated administration may progressively facilitate the expression of spontaneous hindlimb movements.