Environmental factors strongly influence the development of autoimmune diseases, including multiple sclerosis. Despite this clear association, the mechanisms through which environment mediates its effects on disease are poorly understood. Pertussis toxin (PTX) functions as a surrogate for environmental factors to induce animal models of autoimmunity, such as experimental autoimmune encephalomyelitis. Although very little is known about the molecular mechanisms behind its function in disease development, PTX has been hypothesized to facilitate immune cell entry to the CNS by increasing permeability across the blood-brain barrier. Using intravital microscopy of the murine cerebromicrovasculature, we demonstrate that PTX alone induces the recruitment of leukocytes and of active T cells to the CNS. P-selectin expression was induced by PTX, and leukocyte/endothelial interactions could be blocked with a P-selectin-blocking Ab. P-selectin blockade also prevented PTX-induced increase in permeability across the blood-brain barrier. Therefore, permeability is a secondary result of recruitment, rather than the primary mechanism by which PTX induces disease. Most importantly, we show that PTX induces intracellular signals through TLR4, a receptor intimately associated with innate immune mechanisms. We demonstrate that PTX-induced leukocyte recruitment is dependent on TLR4 and give evidence that the disease-inducing mechanisms initiated by PTX are also at least partly dependent on TLR4. We propose that this innate immune pathway is a novel mechanism through which environment can initiate autoimmune disease of the CNS.

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-α production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-α signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.

There has been a great deal of interest in adhesion molecules as targets for the treatment of multiple sclerosis and other inflammatory diseases. In this study, we systematically evaluate α4 integrin and P-selectin as targets for therapy in murine models of multiple sclerosis–for the first time directly measuring the ability of their blockade to inhibit recruitment and relate this to clinical efficacy. Experimental autoimmune encephalomyelitis was induced in C57BL/6 or SJL/J mice and intravital microscopy was used to quantify leukocyte interactions within the CNS microvasculature. In both strains, pretreatment with blocking Abs to either α4 integrin or P-selectin reduced firm adhesion to a similar extent, but did not block it completely. The combination of the Abs was more effective than either Ab alone, although the degree of improvement was more evident in SJL/J mice. Similarly, dual blockade was much more effective at preventing the subsequent accumulation of fluorescently labeled leukocytes in the tissue in both strains. Despite evidence of blockade of leukocyte recruitment mechanisms, no clinical benefit was observed with anti-adhesion molecule treatments or genetic deletion of P-selectin in the C57BL/6 model, or in a pertussis toxin-modified model in SJL/J mice. In contrast, Abs to α4 integrin resulted in a significant delay in the onset of clinical signs of disease in the standard SJL/J model. Despite evidence of a similar ability to block firm adhesion, Abs to P-selectin had no effect. Importantly, combined blockade of both adhesion molecules resulted in significantly better clinical outcome than anti-α4 integrin alone.

Lapointe, Benoît M., Jé rô me Frenette, and Claude H. Cô té . Lengthening contraction-induced inflammation is linked to secondary damage but devoid of neutrophil invasion.

Differences in the regulation of type II glucocorticoid receptor (GR) mRNA levels in female rat brain regions involved in the control of the hypothalamic-pituitary-adrenal axis were studied by Northern blot analysis after chronic administration of corticosterone or dexamethasone to adrenalectomized (ADX), ovariectomized (OVX), and ADX/OVX animals. The effect of chronic estradiol or progesterone treatment of intact animals was also studied. Our results show that type II GR mRNA levels of ADX animals were significantly increased above control values in amygdala (140%) and hippocampus (196%), but not in hypothalamus. These increased transcript levels were down-regulated by corticosterone or dexamethasone, with the exception of those in the amygdala, where corticosterone had no effect. Ovariectomy significantly increased hypothalamic GR mRNA content (174%) over control values, and this increase was sensitive to dexamethasone. The combined effect of adrenalectomy/ovariectomy on GR mRNA levels was greater than that of adrenalectomy only in amygdala. Corticosterone increased amygdala transcript levels in OVX and ADX/OVX animals. Estradiol administration to intact animals raised the GR mRNA content of amygdala, while progesterone treatment had no effect on any of the brain regions studied. We conclude that there exists heterogeneity with respect to type II GR mRNA regulation by corticosterone and dexamethasone in brain regions of ADX female rats, and that certain limbic structures show greater sensitivity to these hormonal manipulations, suggesting a more prominent role in the regulation of the hypothalamic-pituitary-adrenal axis. Our results also suggest that circulating estrogens can influence the sensitivity of brain structures (i.e. hypothalamus and amygdala) to glucocorticoids by altering GR mRNA levels. These regions may represent integration sites at which gonadal steroids are able to alter stress hormone secretion.