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Rationale: Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), are inflammatory disorders of the central nervous system (CNS). The function of platelets in inflammatory and autoimmune pathologies is thus far poorly defined., Objective: We addressed the role of platelets in mediating CNS inflammation in EAE., Methods and Results: We found that platelets were present in human MS lesions as well as in the CNS of mice subjected to EAE but not in the CNS from control nondiseased mice. Platelet depletion at the effector-inflammatory phase of EAE in mice resulted in significantly ameliorated disease development and progression. EAE suppression on platelet depletion was associated with reduced recruitment of leukocytes to the inflamed CNS, as assessed by intravital microscopy, and with a blunted inflammatory response. The platelet-specific receptor glycoprotein Ibα (GPIbα) promotes both platelet adhesion and inflammatory actions of platelets and targeting of GPIbα attenuated EAE in mice. Moreover, targeting another platelet adhesion receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), also reduced EAE severity in mice., Conclusions: Platelets contribute to the pathogenesis of EAE by promoting CNS inflammation. Targeting platelets may therefore represent an important new therapeutic approach for MS treatment.

The aim of this study was to investigate the importance of Toll-like receptor 4 (TLR4) signaling on cardiac myocytes versus immune cells in lipopolysaccharide (LPS)-induced cardiac dysfunction. Cardiac myocytes isolated from LPS-treated C57Bl/6 mice showed reduced shortening and calcium transients as compared with myocytes from untreated mice. In addition, LPS-treated C57Bl/6 mice showed impaired cardiac mitochondrial function, including reduced respiration and reduced time of induction of permeability transition. All of the aforementioned cardiac dysfunction was dependent on TLR4, because LPS-treated TLR4-deficient mice did not have reduced myocyte shortening or mitochondrial dysfunction. To evaluate the role of cardiac myocyte versus leukocyte TLR4, LPS was injected into chimeric mice with TLR4-positive leukocytes and TLR4-deficient myocytes. These mice showed reduced myocyte shortening in response to LPS. Myocytes from chimeric mice with TLR4-deficient leukocytes and TLR4-positive myocytes had no response to LPS. In addition, isolated myocytes from C57Bl/6 mice subsequently treated with LPS and serum for various times did not have reduced shortening, despite the presence of TLR4 mRNA and protein, as determined by reverse-transcription polymerase chain reaction and fluorescent-activated cell sorting. In fact, cardiac myocytes had equivalent amounts of TLR4 as endothelium; however, only the latter is responsive to LPS. Furthermore, signaling pathways downstream of TLR4 were not activated during direct LPS treatment of myocytes. In conclusion, TLR4 on leukocytes, and not on cardiac myocytes, is important for cardiac myocyte impairment during endotoxemia.

In the present study, we investigated whether inhaled nitric oxide (NO) was transported by plasma proteins, such as S-nitroso-albumin (SNO-Alb), in the feline circulation and whether this molecule delivers NO to the periphery under conditions of stress, specifically ischemia/reperfusion (I/R). A flow probe was interposed between the femoral and superior mesenteric artery for blood flow measurements, and a branch of the superior mesenteric vein was cannulated for arterial-venous sampling. In animals breathing room air, SNO-Alb was below detection level in arterial or venous blood. NO inhalation resulted in a significant arterial-venous gradient for SNO-Alb. Concomitant with this loss of SNO-Alb across the intestinal vasculature was an increase in nitrite (NO2-). However, this release of NO was not sufficient to alter intestinal blood flow. I/R during NO inhalation caused a very large increase in arterial SNO-Alb that permitted a 5-fold increase in SNO-Alb consumption and significant generation of NO2- within the postischemic intestinal vasculature. The increased SNO-Alb consumption was sufficient to dramatically improve intestinal blood flow. The very large burst of arterial SNO-Alb during I/R was completely blocked by the administration of superoxide dismutase, suggesting that oxidative stress contributed to the increased SNO-Alb formation. Our data suggest that inhaled NO can increase nitrosothiol production and these molecules may be a functional NO delivery system during cardiovascular disease.

The mechanisms mediating leukocyte recruitment into the cerebral nervous system during inflammation are still poorly understood. The objective of this study was to investigate the leukocyte recruitment in the brain microcirculation by intravital microscopy. Superfusion of the brain with artificial cerebrospinal fluid did not induce leukocyte rolling or adhesion. However, intraperitoneal tumor necrosis factor-alpha (TNF-alpha) caused marked leukocyte rolling and adhesion in the brain microcirculation. Histology revealed that the recruitment was primarily of neutrophils. Both E- and P-selectin were required for TNF-alpha-induced leukocyte recruitment, as rolling was reduced after treatment with either anti-E- or anti-P-selectin antibody and eliminated in E- or P-selectin-deficient mice. A significant increase in brain P- and E-selectin expression was seen after TNF-alpha treatment, but both were an order of magnitude less than in any other tissue. We observed significant platelet paving of TNF-alpha-stimulated endothelium and found that anti-platelet antibody reduced leukocyte rolling and adhesion, as did acetylsalicylic acid (aspirin). However, depletion of platelets did not reduce cerebral P-selectin expression. Moreover, chimeric mice lacking P-selectin on endothelium but not platelets had significantly decreased P-selectin expression and reduced leukocyte recruitment in the brain. This suggests a role for endothelial P-selectin in cerebral leukocyte recruitment. In conclusion, TNF-alpha-induced neutrophil recruitment into the brain requires both endothelial E-selectin and P-selectin as well as platelets, but platelet P-selectin was not a major contributor to this process.

We have previously shown that CD18 and alpha4 integrin were important in the adherence of emigrated neutrophils to cardiac myocytes. Whether either of these molecules is important in myocyte dysfunction is unclear. In this study, we measured contractility as an index of myocyte function. Control contractility was compared with shortening response in myocytes exposed to neutrophils in the presence and absence of anti-CD18 or anti-alpha4 antibodies. Control unloaded cell shortening, expressed as a percentage of resting cell length, measured 10.06+/-1.16% (n=10) at 5 minutes. Circulating neutrophils caused a 35% reduction in cell shortening, an event prevented by anti-CD18, but not by anti-alpha4 antibody. When emigrated neutrophils were added to the myocytes, a profound reduction (50%) in unloaded cell shortening was noted. A significant increase in CD18 and alpha4 integrin was found on emigrated neutrophils. Addition of anti-CD18 antibody did not protect the myocyte from the emigrated neutrophils, whereas the addition of an anti-alpha4 antibody significantly reduced neutrophil-induced cell shortening, despite some neutrophils still adhering to the myocytes. Furthermore, emigrated neutrophils were able to cause myocytes to go into contracture within 5 minutes in the presence of neutrophils with or without anti-CD18 antibody. In addition to the impairment in unloaded cell shortening, at later times (10 minutes), neutrophils also caused a 40% reduction in the rate of contraction and relaxation. The addition of either anti-CD18 or anti-alpha4 antibody protected the myocytes from these changes. The data suggest that immunosuppression of CD18 on emigrated neutrophils was only partially effective in reducing myocyte dysfunction. In contrast, immunosuppression of the alpha4 integrin alone was sufficient to dramatically reduce all parameters of cell dysfunction measured in this study.

The objective of this study was to determine whether endogenous IL-10 is capable of regulating hemodynamic parameters, leukocyte recruitment, and microvascular permeability in response to endotoxin. Intravital microscopy was used to examine hemodynamic parameters, leukocyte rolling and adhesion, and microvascular permeability in cremasteric postcapillary venules in wild-type mice and in IL-10-deficient (IL-10(-/-)) mice exposed to lipopolysaccharide (LPS). Doses of LPS (3 or 30 microg/kg, IV), which did not reduce blood pressure and minimally altered microvascular hemodynamic factors in wild-type mice, caused significant reductions in these parameters in IL-10(-/-) mice, demonstrating at least a 10-fold increased sensitivity in IL-10(-/-) mice to LPS-induced hemodynamic alterations. Furthermore, in response to LPS (30 microg/kg, IV), leukocyte rolling, adhesion, and fluorescein isothiocyanate-albumin extravasation were increased in the IL-10(-/-) mice. Antibody blockade experiments showed that in both types of mice, leukocyte rolling was mediated by E-selectin and P-selectin. Leukocyte accumulation into other tissues, such as lung, also was enhanced greatly in IL-10(-/-) mice. This was specific to endotoxin, because acute chemotactic stimuli including N-formyl-methionyl-leucyl-phenylalanine elicited similar responses in IL-10(-/-) and wild-type mice. These results suggest that endogenous IL-10 may be a homeostatic regulator of hemodynamic parameters, leukocyte-endothelial cell interactions, and microvascular dysfunction in response to endotoxin and provide potential mechanisms to explain the protective effect of IL-10 against LPS-induced mortality.

Previous work has shown that neutrophils isolated from whole blood adhere to cardiac-myocytes via CD18 (beta 2 integrin) to cause injury to the heart cells. In vitro, we have found that upon endothelial transmigration, neutrophils can also express alpha 4 beta 1; however, whether this contributes to neutrophil adhesion to parenchymal cells remains entirely unknown. Unstimulated and tumor necrosis factor-alpha-stimulated rat cardiac myocytes adherent to gelatin-coated coverslips supported N-formyl-Met-Leu-Phe (fMLP)-induced neutrophil (isolated from whole blood) adhesion entirely via CD18 (blocked with monoclonal antibody [mAb] WT-3). Emigrated neutrophils spontaneously adhered to cardiac myocytes also entirely via CD18. However, if fMLP was used to restimulate emigrated neutrophils, the adhesion to cardiac myocytes was entirely independent of CD18. Although an anti-alpha 4 integrin antibody (mAb TA-2) alone did not reduce the emigrated neutrophil-myocyte interaction, dual administration of TA-2 and WT-3 reduced adhesion by 81%. alpha 4 integrin was expressed in small amounts on the surface of circulating neutrophils, increased following transmigration, and then increased > 5-fold after restimulation of these emigrated neutrophils. In the presence of the anti-CD18 antibody, a fibronectin fragment (FN-40) but not a vascular cell adhesion molecule-1 antibody (mAb 5F10) inhibitied neutrophil-myocyte interactions by 80%. Similar results were seen when the rat chemokine CINC-gro was used instead of fMLP, suggesting that the alpha 4-dependent adhesion was not specific to fMLP. These data demonstrate that alpha 4 integrin can be physiologically induced to increase in number and avidity after neutrophil emigration and that this adhesion molecule can cause firm adhesion to fibronectin on parenchymal cells, including rat cardiac myocytes.

Nitric oxide (NO) synthesis inhibition causes neutrophil adhesion to endothelium via a mast cell- and oxidant-dependent mechanism. The objective of this study was to delineate the cascade of events in the mast cell- and oxidant-induced neutrophil-endothelium interactions after NO synthesis inhibition. Mast cells were isolated and purified from the rat peritoneal cavity and coadministered with neutrophils to wells of endothelium. This system was treated with an NO synthesis inhibitor (NG-nitro-L-arginine methyl ester; L-NAME) for 60 minutes. L-NAME did not induce neutrophil-endothelium interactions in the absence of mast cells, but the addition of mast cells in a ratio as low as 1:50 mast cells to neutrophils was sufficient to induce a large increase in neutrophil adhesion to endothelium within 20 to 25 minutes. L-arginine, NO donors, and 8-bromo-cGMP reversed the L-NAME effect, whereas NG-nitro-D-arginine methyl ester alone had no proadhesive effect. The adhesion was inhibited by an anti-CD18 or an anti-intracellular adhesion molecule-1 antibody and a platelet-activating factor-receptor antagonist. Inhibition of NO in isolated endothelial monolayers induced oxidant release (reduction of cytochrome C) into extracellular fluid. The endothelium-derived superoxide contributed to the mast cell-induced adhesion, inasmuch as the extracellular antioxidant superoxide dismutase reduced the neutrophil adhesion response as did disruption of endothelial function. There was some direct activation of mast cells with L-NAME (independent of endothelium) inasmuch as intracellular calcium and oxidative stress increased within mast cells after L-NAME treatment, and this translated into increased neutrophil adhesion to nonendothelial substrata. These data demonstrate that depletion of NO increases oxidative stress within mast cells and endothelium and together these events promote neutrophil adhesion within the vasculature.

The objective of this study was to assess the effect of leukotriene C4 (LTC4) on the flux of rolling leukocytes, leukocyte rolling velocity, and leukocyte adhesion in postcapillary venules in vivo and to study the underlying molecular mechanisms involved. LTC4 (20 nmol/L) induced a rapid and significant increase in leukocyte rolling flux that was inhibitable by an anti-P-selectin antibody and soluble sialyl Lewis(x) (sLe(x)). LTC4 also induced a significant reduction in leukocyte rolling velocity, an event that was independent of P-selectin but entirely dependent on sLe(x). This LTC4-induced reduction in leukocyte rolling velocity was independent of any hemodynamic alterations. Another P-selectin effector, histamine, did not affect leukocyte rolling velocity even at > 5000 times the concentration of LTC4. Treatment with an anti-L-selectin antibody had no effect on the LTC4-induced increase in leukocyte rolling or reduction in rolling velocity. Inhibition of LTC4 bioconversion to LTD4 by pretreatment with L-serine (100 mumol/L) prevented the LTC4-induced increase in leukocyte rolling flux and the LTC4-induced reduction in leukocyte rolling velocity. A subtle, yet significant, increase in leukocyte adhesion was also observed with LTC4. Pretreatment with a platelet-activating factor receptor antagonist returned the LTC4-induced leukocyte rolling velocity to baseline levels. The addition of a very low concentration of platelet-activating factor (1 nmol/L) induced significant leukocyte adhesion in the presence of LTC4 but not histamine.(ABSTRACT TRUNCATED AT 250 WORDS)