The Inflammatory Role Of Fas Ligand In Experimental Stroke And Its Related Pathologic Mechanisms

Background:Stroke is one of the main causes of morbidity and mortality worldwide. Untilnow there is no effective neuroprotectants against stroke damage. Identifying novelneuroprotectants that can protect the injured brain is becoming the most importantevent in the study of cerebrovascular diseases. Inflammation has been shown to be animportant player in the progression of ischemic brain damage. Cerebral inflammationis characterized by the activation of astrocytes and microglia, the accumulation ofinflammatory cells and subsequent production of inflammatory mediators, includingIL-1β, TNF-α, monocyte inflammatory protein-1α (MIP-1α), monocytechemoattractant protein-1(MCP-1) and other deleterious substances. The extent ofinflammatory response has been demonstrated to correlate with the severity of braininjury and long-term outcomes in stroke patients. Understanding the mechanism forpost-stroke inflammation will therefore provide potential targets for stroke management and functional recovery.FasL has been shown to be an apoptosis-inducing factor that triggers cell death insusceptible cells expressing the Fas receptor. In previous studies, FasL had beenthought to be a key component in the establishment of immune privilege. FasLexpression on cancer cells may render them refractory to immune attack and wasso-called “tumor counterattack”. This observation led to the hope that the artificialexpression of FasL on transplanted tissues would act as a local immunosuppressant.Several investigators have sought to mimic this natural immune privilege usingexpression of FasL as a strategy to protect engrafted tissue. However, FasLoverexpression in transplants induced accelerated destruction and rejection of thetransplanted organs, rather than conferring immune privilege. The unexpectedobservations suggested that, besides its pro-apoptotic property, FasL could induceimmuno-inflammatory response. Recently, FasL was suggested to be capable ofactivating signal transduction pathways that induce inflammatory responses. It hasbeen shown that FasL induces the production of pro-inflammatory cytokines andchemokines including IL-6, MCP-1and IL-8in various cells types, and recruitneutrophil and/or T lymphocyte accumulation. Meanwhile FasL is also regarded as anaccessory or costimulatory molecule for T cells activation. In fact, FasL has beeninvolved in many inflammatory response such as lung, heart and et al. FasL mutationcould significantly improve the function of these organs. Therefore, FasL plays ancritical role in the immuno-inflammatory system.In central nervous system (CNS), FasL have been observed to be elevated incompromised brains in a variety of neurological disorders including brain trauma,ischemia stress and et al. The inflammatory role of FasL in ischemic stroke, however,is not fully understood. Most previous studies have associated the effect of FasL inischemic brain injury solely with its well-known pro-apoptotic property. Accepting that inflammatory reaction is one of the most important mechanisms after stroke, andFasL is considered a major modulator in the immune and inflammatory response, wemight ask the following questions:1) Does FasL exert an inflammatory effect in theonset and development of experimental stroke?2) Could FasL be able to induceinflammatory response directly by itself and independent of cell apoptosis?3）Howdose FasL promote inflammation during these processes, and which intracellularnon-apoptotic signaling pathways are triggered? Both of these important questions arecurrently unresolved and are desired further investigations.PART I FasL induces brain inflammatory in experimental strokeand exacerbates brain damageAims: To determine the possible role of FasL in inducing inflammatory responseafter cerebral ischemia in mice.Materials and Methods: Twelve to fourteen-week-old male FasL mutant (gld)mice (22–24g) and their wild type control C57BL/6J mice (B6) were chosen asexperimental animals. Focal cerebral ischemia was induced by transient middlecerebral artery occlusion (MCAO) operation for2h. The reperfusion time windowswere6h,24h and72h. The sensorimotor dysfunction was assessed by neurologicalseverity score (NSS), the infarct volume was measured by TTC staining and brainedema was obtained by water content formula. Real-time PCR (qPCR) was used todetect mRNA expressions of inflammatory cytokines (IL-1β, TNF-α, IFN-γ),anti-inflammatory cytokines (IL-4, IL-10) and chemokines (MCP-1, MIP-1α, MIP-2).Protein levels of IL-1β and TNF-α were detected by enzyme-linked immunosorbentassay (ELISA) in both ischemic cortex and blood serum. Immunostaining were usedto observe the expression of GFAP+, Iba1+, MPO+and CD3+in ischemic hemisphere.The T lymphocytes subpopulations CD4+, CD8+, Th1/Th2were assayed by flow cytometry (FACS) in the ischemic brain and peripheral blood.Results:1) Both B6and gld mice showed neurological deficits and infarctvolumes from6h to72h time windows after ischemic stroke. Severe neurologicaldeficits and larger volumes were observed in B6mice. FasL mutation profoundlyimproved neurological performance and reduced brain edema and infarct volumes.2)The mRNA expressions of pro-infammatory cytokines (IL-1β, TNF-α, IFN-γ) andchemokines (MCP-1, MIP-1α, MIP-2) were attenuated in ischemic cortex of gld miceat24h after ischemia reperfusion. In contrast, the level of anti-inflammatory factorIL-10was up-regulated in gld mice, while the level of IL-4showed no significantchange between the two genotypes. At24h after cerebral ischemia, FasL mutationinhibited the activation of residential glial cells (Iba1+microglia and GFAP+astrocytes) and attenuated the recruitment of peripheral ifnammatory cells (MPO+neutrophils) into CNS. Although the total numbers of T cells infiltration are similar inboth gld and B6mice, the distributions of T cell subsets were different. FACSanalysis showed the infiltration of both CD3+CD4+and CD3+CD8+T cells into thebrain at24h after MCAO. Mutation of FasL in gld mice resulted in reducedinfiltration of CD8+T cells and turned the Th1/Th2balance towards Th2both in thebrain and peripheral blood after cerebral ischemia.Conclusion: FasL exerts an inflammatory effect after stroke, FasL mutationcould inhibit inflammation and protect ischemia injury.1) FasL mutation reducespro-inflammatory cytokines and chemokines in ischemic brain.2) FasL mutationattenuates the cellular inflammatory reaction (astrocytes and microglia activation andneutrophils infiltration) associated with cerebral ischemia.3) FasL mutationmodulates T lymphocyte subsets (decreases CD8+T cell and CD4+Th1/Th2ratio)after cerebral ischemia. Part II FasL contributes to inflammatory responseindependent of cell apoptosisAims: To prove that FasL could directly induce inflammation after cerebralischemia without significant cell apoptosis in FasL mutate gld mice.Materials and Methods: We used intraperitoneal administration ofLipopolysaccharide (LPS i.p,0.5mg/kg)24h before MCAO in B6mice in order toprotect ischemia injury that similar to gld mice. The infarct volume and neutrophilinfiltration of the three groups mice (B6, gld, pretreatment of B6mice with LPS)were compared at24h after MCAO. Annexin V staining were used as a probe todetect apoptosis. Double immunofluorescence was used for identifying the apoptoticinflammatory cells (astrocytes, microglia, neutrophils, T cells) and apoptotic neurons.Then we used intracerebraventricular (ICV) LPS injection in a dose of1ug to inducebrain inflammation in B6and gld mice. Real-time PCR was used to detect mRNAexpressions of inflammatory cytokines and chemokines. Immunofluorescence wasused for apoptotic cells detection and immunohistochemical staining was taken forobservation of morphological changes and expressions of astrocytes, microglia andneutrophils in LPS-induced brain inflammation in B6and gld mice (without MCAO).Results:1) As the intensity of the inflammatory reaction is related to the size ofischemic lesion, and small infarcts should have less inflammation than large infarcts.It is possible that the attenuation in brain inflammation in gld mice could be aconsequence of the reduced infarct size. To address this, we took an alternativestrategy. Pretreatment of B6mice with LPS (0.5mg/kg)24h before MCAO could leadto a reduced infarct volume that was similar in size to that seen in gld mice, but B6mice pretreatment of LPS had more neutrophil infiltration compared to gld mice. Todetermine whether the reduction in neutrophil infiltraiton in gld mice wascommensurate with the reduction in infarct size, a linear regression analysis was performed between the number of neutrophils at each rostrocaudal level and thecorresponding infarct area. In B6mice pretreatment of LPS, the slope of theregression line overlapped with that of untreated B6mice, indicating that thereduction in the number of neutrophils was proportional to the reduction in infarctsize. In contrast, the slope of the regression line in gld mice was lower than that of B6mice, indicating that the reduction in neutrophils was greater than anticipated fromthe smaller size of the infarct volume.2) As the process of cerebral ischemia also associated with cell apoptosis, it ispossible that the attenuated inflammatory reaction in gld mice was a consequence ofdecreased cell apoptosis, especially neurons. To address this issue, we used AnnexinV as a probe for detecting early apoptosis. The lacking colocalization of Iba1, GFAP,MPO, CD3with Annexin V staining indicated that there was little apoptosis ofmicroglia, astrocytes, neutrophils or CD3+T cells in both the B6and gld brains afterMCAO. We further used neuronal nuclear antigen (NeuN) as a probe for detectingsurvival neurons, and the gld mice showed less neuron apoptosis than B6mice at24hafter MCAO.3) Ischemic insult obviously increased neuron apoptosis in B6mice while gldmutation could partially abrogate this effect. In order to exclude the possibility thatless inflammatory response observed in gld mice after MCAO was simply due toreduced apoptotic loss of neurons, we took another inflammation model of LPSinjection (ICV,1ug) without MCAO to show that FasL had a direct role oninflammation. Both literature and our results confirmed that ICV LPS of small dose(1ug) could induce brain inflammation without triggering obvious neuron apoptosis.In contrast to cerebral ischemia, the molecular and cellular finammatory changesinduced by ICV LPS were also attenuated in gld mice.Conclusion: FasL could directly exert an immuno-inflammatory effect in CNS after ischemic stroke, which is independent of cell apoptosis.1) In FasL mutate gld miceneutrohil infiltration was suppressed more than expected on the basis of the reductionin infarct size.2) FasL mutation attenuated cerebral inflammation independent ofinflammatory cells apoptosis in CNS after ischemic stroke.3) FasL mutationinhibited LPS induced inflammation in brain without being affected by apoptoticneuron death.Part III The pathologic mechanisms of FasL mutation inpost-stroke inflammatory responseAims: To investigate the intracellular molecular mechanisms and signalpathways of FasL in modulating inflammatory response associated with cerebralischemia and find the potential target for stroke treatment.Materials and Methods: To detect sFasL, cytosolic protein fractions from braincortex were prepared using ultracentrifuge at100,000g for60min. Total proteins ofcortex were also extracted to detect phospho-SAPK/JNK, phospho-p38,phospho-p44/42. The protein expression of sFasL and activation of MAPK signalpathways (JNK, ERK and p38) after MCAO were detected by western blot. Doubleimmunofluorescence was used to examine the Fas expression in astrocytes aftercerebral ischemia.Results:1) In the ischemic cortex of B6mice, the level of sFasL increased by451.67%over the sham-operated brain. The expression of sFasL decreased by62.7%in the gld cortex as compared to B6mice. These results suggested that gld mice hadless sFasL than B6mice that might contribute to its less inflammatory function afterMCAO.2) The activation of MAPK signal pathways (JNK, ERK and p38) has beenshown to contribute to cerebral inflammation in ischemic injury. Our study revealed that phosphorylation of JNK and ERK increased significantly in ischemic cortex ofB6mice. FasL mutation inhibited the phosphorylation of JNK, but did not affect thephosphorylation of p38or ERK.3) Our results suggested that JNK signaling might beinvolved in FasL induced inflammatory reaction after stroke, and JNK pathway wasalso reported to be very important in inducing FasL reverse signaling of T cellsactivation and proliferation. At the beginning of ischemia, T cells infiltration intobrain might encounter the activated astrocytes first, and astrocytes might be the mostsignificant antigen-presenting cell (APC) cells in CNS that express Fas protein. So wenext detected the Fas expression in astrocytes after ischemia, and the results showedthat B6mice had significantly increased Fas level in GFAP+astrocytes compared toB6mice. Our study revealed that the high level of Fas in GFAP+astrocytes mightactivate the FasL reverse signaling and contribute to T cells migration andproliferation, thus exacerbating the immuno-inflammatory response after ischemiastroke.Conclusion:1) FasL plays an infammatory role in cerebral ischemia. Themechanisms might relate to sFasL and the c-Jun N-terminal kinase (JNK) signalingpathway.2) The high expression of Fas in GFAP+astrocytes might activate the FasLreverse signaling and contribute to T cells migration and proliferation after cerebralischemia3) Neutralization of FasL may be a novel therapeutic strategy to suppresspost-stroke infammation and improve the long-term outcomes of stroke.In summary, the novel findings of this study are as follows:1. FasL is an important player in ischemic brain injury after stroke due to its potencyto provoke inflammation.2. FasL could contribute to post-ischemia injury through activating residential CNS inflammatory cells, recruiting peripheral inflammatory cells, and modulating Tcell subpopulations.3. sFasL and JNK signaling might contribute to FasL induced inflammatoryfunction.