RESUMO
High-grade gliomas harbor abundant myeloid cells that suppress anti-tumor immunity and support tumor growth. Targeting transcription factors, such as NF-κB p50, that mediate suppressive myeloid M2 polarization may prove therapeutic. GL261-Luc glioblastoma cells were inoculated into wild-type and p50-/- mice, followed by analysis of tumor growth, survival, tumor myeloid cells, and T cells. The absence of host p50 slows tumor growth and enables regression in 30% of recipients, leading to prolonged survival. Tumors developing in p50-/- mice possess a greater concentration of tumor-infiltrating myeloid cells (TIMs) than those in wild-type mice. TIMs are predominantly F4/80hi macrophages which, along with tumor-associated microglia, express increased pro-inflammatory M1 and reduced immune-suppressive M2 markers. In p50-/- mice, total tumor CD4 T cells are threefold more abundant, whereas CD8 T-cell numbers are unchanged, and both produce increased IFNγ and Granzyme B. Naïve splenic p50-/- CD8 T cells manifest increased activation, whereas naïve p50-/- and WT CD4 T cells show similar Th1, Th2, and Th17 polarization. Antibody targeting CD4, but not CD8, fully obviates the p50-/- survival advantage. Combined CD4 and CD8 T-cell depletion reverses myeloid M2 polarization in wild-type hosts, without affecting myeloid M1 polarization in p50-/- hosts. Finally, gliomas grow similarly in p50(f/f) and p50(f/f);Lysozyme-Cre mice, the latter having reduced p50 specifically in myeloid cells and tumor microglia. Thus, high-grade glioma T cells play a key role in directing M2 polarization of tumor myeloid cells, and reducing NF-κB p50 in both tumor myeloid cells and T cells may contribute to glioma therapy.
Assuntos
Glioblastoma/prevenção & controle , Macrófagos/imunologia , Células Mieloides/imunologia , Subunidade p50 de NF-kappa B/fisiologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Glioblastoma/imunologia , Glioblastoma/mortalidade , Ativação Linfocitária , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Taxa de Sobrevida , Linfócitos T/metabolismoRESUMO
Astrocytes undergo rapid activation after injury, which is mediated in part by the transcription factor nuclear factor-kappaB (NF-κB). Consequently, activated astrocytes have been shown to induce the NF-κB regulated phagocyte NADPH oxidase (PHOX), resulting in elevated production of reactive oxygen species. We investigated the regulatory mechanisms of PHOX-induced oxidative stress in astrocytes and its non-cell-autonomous effects on retinal ganglion cell loss following retinal ischemia-reperfusion (IR) injury. To study PHOX activity and neurotoxicity mediated by glial NF-κB, we employed GFAP-IκBα-dn transgenic mice, where the NF-κB canonical pathway is suppressed specifically in astrocytes. Our analysis showed that NF-κB activation in astrocytes correlated with an increased expression of PHOX and reactive oxygen species production in primary cells and whole retinas subjected to oxygen-glucose deprivation or IR injury. Selective blockade of NF-κB in astrocytes or application of NADPH oxidase inhibitors suppressed retinal ganglion cell loss in co-cultures with astroglia challenged by oxygen-glucose deprivation. Furthermore, genetic suppression of astroglial NF-κB reduced oxidative stress in ganglion layer neurons in vivo in retinal IR. Collectively, our results suggest that astroglial NF-κB-regulated PHOX activity is a crucial toxicity pathway in the pathogenesis of retinal IR injury.
Assuntos
Astrócitos/metabolismo , NADPH Oxidases/metabolismo , NF-kappa B/fisiologia , Estresse Oxidativo , Traumatismo por Reperfusão/metabolismo , Doenças Retinianas/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/enzimologia , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NADPH Oxidases/fisiologia , Estresse Oxidativo/genética , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Doenças Retinianas/enzimologia , Doenças Retinianas/genéticaRESUMO
PURPOSE: We investigated the effect of ATP (ATP) encapsulated in liposomes (ATP-liposomes) on the level of inflammation and neuronal death in the retina induced by ischemia reperfusion (IR). METHODS: Primary retinal ganglion cells treated with ATP-liposomes, empty liposomes, and phosphate buffer solution (PBS) were deprived of oxygen and glucose (OGD) for 6 h in vitro, in an anaerobic chamber. Plates were assessed for the proportion of necrotic versus apoptotic cells and for cell survival 12 h after OGD. For in vivo experiments, we induced retinal ischemia by unilateral elevation of intraocular pressure for 1 h by direct corneal canulation. Mice were injected with liposomes or PBS 24 h before IR, at the time of surgery, and every 24 h until sacrifice. Transmission electron microscopic analysis was used to identify necrotic and apoptotic cells in ischemic retinas. The changes in expression of pro-inflammatory genes 24 h post reperfusion were assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Corresponding changes in protein abundances were analyzed by immunohistochemistry. Cell death was evaluated by direct counting of neurons in the ganglion cell layer (GCL) of flatmounted retinas 7 days post reperfusion. RESULTS: Treatment with ATP-liposomes increases retinal ganglion cell (RGC) survival and decreases necrotic cell death following OGD. Injection of ATP-liposomes markedly decreased necrotic cell death in the GCL following retinal ischemia. The ATP-liposome treatment reduced the expression of pro-inflammatory genes, including that of interleukin 1ß (Il1ß), interleukin 6 (Il6), tumor necrosis factor (Thf), chemokine (C-C motif) ligand 2 (Ccl2), chemokine (C-C motif) ligand 5 (Ccl5), chemokine (C-X-C motif) ligand 10 (Cxcl10), intercellular adhesion molecule 1 (Icam1), and nitric oxide synthase 2 (Nos2), in the retina 24 h after IR and significantly reduced the GCL neuron death rate 7 days after reperfusion. CONCLUSIONS: ATP-liposome treatment of IR-challenged neural tissues suppressed necrosis and correlated with a significantly reduced level of inflammation and retinal damage.
Assuntos
Trifosfato de Adenosina/administração & dosagem , Trifosfato de Adenosina/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , Retina/patologia , Trifosfato de Adenosina/farmacologia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Barreira Hematorretiniana/patologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/deficiência , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/patologia , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio , Traumatismo por Reperfusão/complicações , Retina/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/ultraestrutura , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/metabolismo , Neurônios Retinianos/patologiaRESUMO
PURPOSE: We investigated whether retinal ischemia and inflammation produced by raising the intraocular pressure above normal systolic levels differs in mice that lack a functional toll-like receptor 4 (Tlr4) signaling pathway. METHODS: In this work we used the murine strain B6.B10ScN-Tlr4(lps-del)/JthJ, which does not express functional Tlr4. C57BL/6J was considered as the control. We induced retinal ischemia by unilateral elevation of intraocular pressure for 1 h by direct corneal cannulation. The changes in expression of proinflammatory genes 24 h postreperfusion were assessed by quantitative PCR. Corresponding changes in protein abundances were analyzed by western blot and immunohistochemistry. Cell death was evaluated by direct counting of neurons in the ganglion cell layer of flat-mounted retinas seven days postreperfusion. RESULTS: We showed that Tlr4-deficient mice display significantly reduced expression of proinflammatory genes, including RelA, tumor necrosis factor (Thf), interleukin 6 (Il6), chemokine (C-C motif) ligand 2 (Ccl2), chemokine (C-C motif) ligand 5 (Ccl5), chemokine (C-X-C motif) ligand 10 (Cxcl10), Cybb, nitric oxide synthase 2 (Nos2), and intercellular adhesion molecule 1 (Icam1) 24 h after reperfusion. The mice that lacked Tlr4 showed significantly increased survival of neurons in the ganglion cell layer following ischemic injury, as compared to wild-type controls. CONCLUSIONS: Our results indicate that Tlr4 signaling is involved in retinal damage and inflammation triggered by ischemic injury.
Assuntos
Traumatismo por Reperfusão/metabolismo , Retina/metabolismo , Retina/patologia , Receptor 4 Toll-Like/metabolismo , Animais , Sobrevivência Celular , Citoproteção , Inflamação/complicações , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologia , Neurônios Retinianos/metabolismo , Neurônios Retinianos/patologia , Transdução de Sinais , Receptor 4 Toll-Like/deficiênciaRESUMO
BACKGROUND: Macrophages and dendritic cells lacking the transcription factor nuclear factor kappa B p50 are skewed toward a proinflammatory phenotype, with increased cytokine expression and enhanced T cell activation; additionally, murine melanoma, fibrosarcoma, colon carcinoma, and glioblastoma grow slower in p50-/- mice. We therefore evaluated the efficacy of p50-negative immature myeloid cells (p50-IMCs) adoptively transferred into tumor-bearing hosts. Immature cells were used to maximize tumor localization, and pretreatment with 5-fluorouracil (5FU) was examined due to its potential to impair marrow production of myeloid cells, to target tumor myeloid cells and to release tumor neoantigens. METHODS: Wild-type (WT)-IMC or p50-IMC were generated by culturing lineage-negative marrow cells from WT or p50-/- mice in media containing thrombopoietin, stem cell factor and Flt3 ligand for 6 days followed by monocyte colony-stimulating factor for 1 day on ultralow attachment plates. Mice inoculated with Hi-Myc prostate cancer (PCa) cells or K-RasG12D pancreatic ductal carcinoma (PDC)-luciferase cells received 5FU followed 5 days later by three doses of 107 immature myeloid cells (IMC) every 3-4 days. RESULTS: PCa cells grew slower in p50-/- mice, and absence of host p50 prolonged the survival of mice inoculated orthotopically with PDC cells. IMC from Cytomegalovirus (CMV)-luciferase mice localized to tumor, nodes, spleen, marrow, and lung. 5FU followed by p50-IMC slowed PCa and PDC tumor growth, ~3-fold on average, in contrast to 5FU followed by WT-IMC, 5FU alone or p50-IMC alone. Slowed tumor growth was evident for 93% of PCa but only 53% of PDC tumors; we therefore focused on PCa for additional IMC analyses. In PCa, p50-IMC matured into F4/80+ macrophages, as well as CD11b+F4/80-CD11c+ conventional dendritic cells (cDCs). In both tumor and draining lymph nodes, p50-IMC generated more macrophages and cDCs than WT-IMC. Activated tumor CD8+ T cells were increased fivefold by p50-IMC compared with WT-IMC, and antibody-mediated CD8+ T cell depletion obviated slower tumor growth induced by 5FU followed by p50-IMC. CONCLUSIONS: 5FU followed by p50-IMC slows the growth of murine prostate and pancreatic carcinoma and depends on CD8+ T cell activation. Deletion of p50 in patient-derived marrow CD34+ cells and subsequent production of IMC for adoptive transfer may contribute to the therapy of these and additional cancers.
Assuntos
Carcinoma Ductal Pancreático/terapia , Imunoterapia Adotiva/métodos , Células Mieloides/imunologia , Células Mieloides/transplante , Subunidade p50 de NF-kappa B/deficiência , Neoplasias Pancreáticas/terapia , Neoplasias da Próstata/terapia , Animais , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Feminino , Fluoruracila/farmacologia , Masculino , Camundongos , Subunidade p50 de NF-kappa B/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologiaRESUMO
Different sub-populations of retinal ganglion cells (RGCs) vary in their sensitivity to pathological conditions such as retinal ischemia, diabetic retinopathy and glaucoma. Comparative transcriptomic analysis of such groups will likely reveal molecular determinants of differential sensitivity to stress. However, gene expression profiling of primary neuronal sub-populations represent a challenge due to the cellular heterogeneity of retinal tissue. In this manuscript, we report the use of a fluorescent neural tracer to specifically label and selectively isolate RGCs with different soma sizes by fluorescence-activated cell sorting (FACS) for the purpose of differential gene expression profiling. We identified 145 genes that were more active in the large RGCs and 312 genes in the small RGCs. Differential data were validated by quantitative RT-PCR, several corresponding proteins were confirmed by immunohistochemistry. Functional characterization revealed differential activity of genes implicated in synaptic transmission, neurotransmitter secretion, axon guidance, chemotaxis, ion transport and tolerance to stress. An in silico reconstruction of cellular networks suggested that differences in pathway activity between the two sub-populations of RGCs are controlled by networks interconnected by SP-1, Erk2 (MAPK1), Egr1, Egr2 and, potentially, regulated via transcription factors C/EBPbeta, HSF1, STAT1- and c-Myc. The results show that FACS-aided purification of retrogradely labeled cells can be effectively utilized for transcriptional profiling of adult retinal neurons.
Assuntos
Perfilação da Expressão Gênica/métodos , Proteínas do Tecido Nervoso/genética , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Separação Celular , Tamanho Celular , Sobrevivência Celular/genética , Citometria de Fluxo/métodos , Corantes Fluorescentes , Imuno-Histoquímica , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Long-Evans , Células Ganglionares da Retina/classificação , Células Ganglionares da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional/genéticaRESUMO
The microenvironment of prostate cancer often includes abundant tumor-associated macrophages (TAMs), with their acquisition of an M2 phenotype correlating with local aggressiveness and metastasis. Tumor-derived M-CSF contributes to TAM M2 polarization, and M-CSF receptor inhibition slows prostate cancer growth in model systems. As additional cytokines can direct TAM M2 polarization, targeting downstream transcription factors could avoid resistance. Klf4 and C/EBPß each contribute to monocyte development, and reduced expression of macrophage Klf4 or C/EBPß favors their adoption of a pro-inflammatory M1 state. We find that a Hi-Myc C57BL/6 prostate cancer line grows more slowly in syngeneic Klf4(f/f);Lys-Cre compared with Klf4(f/f) mice when inoculated subcutaneously, but grows equally rapidly in C/EBPß(f/f);Lys-Cre and C/EBPß(f/f) hosts. In the absence of myeloid Klf4, TAMs have reduced expression of surface mannose receptor and Fizz1 mRNA, both M2 markers. Global gene expression analysis further revealed activation of pro-inflammatory, pro-atherosclerotic pathways. Analysis of tumor-infiltrating lymphocytes (TILs) demonstrated markedly increased activated CD8 T cell numbers, and CD8 T cell depletion obviated the inhibitory effect of myeloid Klf4 deletion on prostate cancer growth. These findings suggest that reducing expression or activity of the Klf4 transcription factor in tumor myeloid cells may contribute to prostate cancer therapy.
Assuntos
Fatores de Transcrição Kruppel-Like/deficiência , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Animais , Aterosclerose/etiologia , Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Proteína beta Intensificadora de Ligação a CCAAT/genética , Antígeno CD11c/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Lectinas Tipo C/metabolismo , Linfócitos do Interstício Tumoral , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Neoplasias da Próstata/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptores de Superfície Celular/metabolismo , Microambiente TumoralRESUMO
The purpose of this study was to ascertain the mechanisms by which advanced prostate cancer cells resist bortezomib therapy. Several independent studies have shown that cells are protected from proteasome inhibition by increased autophagic activity. We investigated whether C/EBPß, a transcription factor involved in the control of autophagic gene expression, regulates resistance to proteasome inhibition. In PC3 cells over-expressing C/EBPß, turnover of autophagic substrates and expression of core autophagy genes were increased. Conversely, C/EBPß knockdown suppressed autophagosome-lysosome fusion. We also found that C/EBPß knockdown suppressed REDD1 expression to delay early autophagy, an effect rescued by exogenous REDD1. Cells with suppressed C/EBPß levels showed delayed autophagy activation upon bortezomib treatment. Knockdown of C/EBPß sensitized PC3 cells to bortezomib, and blockade of autophagy by chloroquine did not further increase cell death in cells expressing shRNA targeting C/EBPß. Lastly, we observed a decreased growth of PC3 cells and xenografts with C/EBPß knockdown and such xenografts were sensitized to bortezomib treatment. Our results demonstrate that C/EBPß is a critical effector of autophagy via regulation of autolysosome formation and promotes resistance to proteasome inhibitor treatment by increasing autophagy.
Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lisossomos/metabolismo , Masculino , Fusão de Membrana , Camundongos Endogâmicos NOD , Camundongos SCID , Fagossomos/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Ligação Proteica , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Following spinal cord injury (SCI), both an inhibitory environment and lack of intrinsic growth capacity impede axonal regeneration. In a previous study, prevention of cyclic adenosine monophosphate (AMP) hydrolysis by the phosphodiesterase-4 inhibitor rolipram, in combination with Schwann cell (SC) grafts, promoted significant supraspinal and proprioceptive fiber growth and/or sparing and improved locomotion. In another study, transplanted SCs transduced to generate a bifunctional neurotrophin (D15A) led to significant increases in graft SCs and axons, including supraspinal and myelinated axons. Here we studied the growth and myelination of local and supraspinal axons and functional outcome following the combination of rolipram administration and neurotrophin-transduced SC implantation after SCI. Rolipram was administered subcutaneously for 4 weeks immediately after contusion at vertebral T8 (25.0-mm weight drop, MASCIS impactor). GFP or GFP-D15A-transduced SCs were injected into the injury epicenter 1 week after SCI. GFP-D15A SC grafts and GFP SC grafts with rolipram contained significantly more serotonergic fibers compared to GFP SCs. SC myelinated axons were increased significantly in GFP SC with rolipram-treated animals compared to animals receiving SCI alone. Rolipram administered with either GFP or GFP-D15A SCs significantly increased numbers of brain stem-derived axons below the lesion/implant area and improved hindlimb function. Compared to the single treatments, the combination led to the largest SC grafts, the highest numbers of serotonergic fibers in the grafts, and increased numbers of axons from the reticular formation below the lesion/implant area and provided the greatest improvement in hindlimb function. These findings demonstrate the therapeutic potential for a combination therapy involving the maintenance of cyclic AMP levels and neurotrophin-transduced SCs to repair the subacutely injured spinal cord.
Assuntos
Antidepressivos/uso terapêutico , Neurotrofina 3/metabolismo , Rolipram/uso terapêutico , Células de Schwann/transplante , Traumatismos da Medula Espinal/cirurgia , Animais , Axônios/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membro Posterior/fisiologia , Atividade Motora , Bainha de Mielina/metabolismo , Neurotrofina 3/genética , Ratos , Ratos Endogâmicos F344 , Recuperação de Função Fisiológica , Regeneração , Células de Schwann/citologia , Células de Schwann/metabolismo , Serotonina/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologiaRESUMO
The implantation of exogenous cells or tissues has been a popular and successful strategy to overcome physical discontinuity and support axon growth in experimental models of spinal cord injury (SCI). Cellular therapies exhibit a multifarious potential for SCI restoration, providing not only a supportive substrate upon which axons can traverse the injury site, but also reducing progressive tissue damage and scarring, facilitating remyelination repair, and acting as a source for replacing and re-establishing lost neural tissue and its circuitry. The past two decades of research into cell therapies for SCI repair have seen the progressive evolution from whole tissue strategies, such as peripheral nerve grafts, to the use of specific, purified cell types from a diverse range of sources and, recently, to the employment of stem or neural precursor cell populations that have the potential to form a full complement of neural cell types. Although the progression of cell therapies from laboratory to clinical implementation has been slow, human SCI safety and efficacy trials involving several cell types within the US appear to be close at hand.
Assuntos
Transplante de Medula Óssea/métodos , Transplante de Células/métodos , Traumatismos da Medula Espinal/terapia , Adulto , Animais , Transplante de Medula Óssea/tendências , Transplante de Células/tendências , Humanos , Coleta de Tecidos e Órgãos/métodosRESUMO
Microglia respond in a stereotypical pattern to a diverse array of pathological states. These changes are coupled to morphological and immunophenotypical alterations and the release of a variety of reactive species, trophic factors and cytokines that modify both microglia and their cellular environment. We examined whether a microglial-produced cytokine, tumour necrosis factor-alpha (TNF-alpha), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF-alpha antisense deoxyoligonucleotides (ASOs). Microglia and macrophages harvested from 3 d post-contused rat spinal cord were large and rounded (86.3 +/- 9.6%). They were GSA-IB4-positive (GSA-IB4(+)) (Griffonia simplicifolia lectin, microglia specific; 94.8 +/- 5.1%), strongly OX-42 positive (raised against a type 3 complement/integrin receptor, CD11b; 78.9 +/- 9.1%), ED-1 positive (a lysosomal marker shown to correlate well with immune cell activation; 97.2 +/- 2.6%) and IIA positive (antibody recognizes major histocompatibility complex II; 57.2 +/- 5.6%), indicative of fully activated cells, for up to 48 h after plating. These cells also secreted significant amounts of TNF-alpha (up to 436 pg/microg total protein, 16 h). Fluoroscein isothiocyanate-labelled TNF-alpha ASOs (5, 50 and 200 nm) added to the culture medium were taken up very efficiently into the cells (> 90% cells) and significantly reduced TNF-alpha production by up to 92% (26.5 pg/microg total protein, 16 h, 200 nm TNF-alpha ASOs). Furthermore, few of the treated cells at this time were round (5.4 +/- 2.7%), having become predominantly spindle shaped (74.9 +/- 6.3%) or stellate (21.4 +/- 2.7%); immunophenotypically, although all of them remained GSA-IB4 positive (91.6 +/- 6.2%), many were weakly OX-42 positive and few expressed either ED-1 (12.9 +/- 2.5%) or IIA (19.8 +/- 7.4%). Thus, the secretion of TNF-alpha early in spinal cord injury may be involved in autoactivating microglia/macrophages. However, at the peak of microglial activation after injury, the activation state of microglia/macrophages is not stable and this process may still be reversible by blocking TNF-alpha.