Tumor necrosis factor identified in multiple sclerosis brain.

Frozen brain specimens from patients with multiple sclerosis (MS) and other neurologic diseases were analyzed using immunocytochemical techniques for the presence of TNF. In brain lesions in MS, and subacute sclerosing panencephalitis, TNF+ cells were demonstrated. At the lesion site in MS, TNF+ staining is associated with both astrocytes and macrophages. These observations were not made in Alzheimer's disease or normal brain tissue. The presence of TNF in MS lesions suggests a significant role for cytokines and the immune response in disease progression.

Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript.

Latent infections with periodic reactivation are a common outcome after acute infection with many viruses. The latency-associated transcript (LAT) gene is required for wild-type reactivation of herpes simplex virus (HSV). However, the underlying mechanisms remain unclear. In rabbit trigeminal ganglia, extensive apoptosis occurred with LAT(-) virus but not with LAT(+) viruses. In addition, a plasmid expressing LAT blocked apoptosis in cultured cells. Thus, LAT promotes neuronal survival after HSV-1 infection by reducing apoptosis.

Expression of HLA-DR antigens in human fetal pancreas tissue.

The expression of HLA-DR antigens in human fetal pancreas tissue was studied using four independently derived monoclonal antibodies recognizing nonpolymorphic HLA-DR antigens. Immunoperoxidase staining of frozen sections revealed numerous DR-antigen-positive cells. Two major cell types could be distinguished. Large, strongly DR-positive cells with dendritic morphology were distributed throughout the dendritic morphology were distributed throughout the pancreatic parenchyma and interstitial connective tissue. Dual staining of DR antigens and insulin using a two-color immunoperoxidase technique clearly showed that some of these DR-positive dendritic cells were located within and in close association with insulin-producing islets. Also observed as a regular feature of fetal pancreas tissue sections were large clusters of DR-positive cells with lymphoid morphology. Positive staining of many cells within these clusters with monoclonal antibodies against T-cell marker (Leu-1) and T-cell subset markers (Leu-2 and -3) supported their classification as lymphoid cells. In contrast, no positive staining with anti-T-cell antibodies was observed outside of lymphoid cell clusters, confirming the distinction between these cells and the DR-positive cells distributed throughout the pancreas. The presence of other DR-bearing cell types in fetal pancreas could not be excluded, but most endothelial cells appeared to be DR-negative. The demonstration of numerous DR-bearing cells associated with fetal pancreas tissue may require that we alter our views on DR-antigen ontogeny and develop new strategies for fetal pancreas transplantation.

Soluble TNF-alpha receptors are constitutively shed and downregulate adhesion molecule expression in malignant gliomas.

The regulation of adhesion molecule expression in malignant gliomas by tumor necrosis factor-alpha (TNF) and soluble TNF receptors (TNFR) was examined in the malignant glioma cell line A-172 and in 2 primary glioblastoma cell cultures (LA-492 and LA-567). A-172 cells expressed only the p55 TNF receptor transcripts and protein. The 2 primary cell cultures expressed both the p55 and p75 TNF receptors. In A-172 cells and in 1 of 2 primary glioma cell cultures, TNF upregulated the expression of ICAM-1 and VCAM-1, A-172 and both primary glioma cultures also shed their TNF receptors in the absence of activation by stimulating agents. Soluble p55 (sp55) receptors, but not soluble p75 (sp75) receptors, were found to reduce the TNF induced VCAM-1 and ICAM-1 expression in both the glioma cell line and the primary cell culture. Immunostaining of malignant glioma sections confirmed the presence of soluble TNFR and adhesion molecule expression in glioma cells in situ. These data suggest that soluble TNF receptors may play a role in the mechanism by which malignant gliomas downregulate the effects of infiltrating immune-competent cells.

Circulating antibody against tumor necrosis factor-alpha protects rat brain from reperfusion injury.

The role of tumor necrosis factor-alpha (TNF alpha) in brain injury is controversial. We studied the effect of anti-TNF-alpha antibody in a rat model of reversible middle cerebral artery occlusion. During focal ischemia and early reperfusion, TNF-alpha was rapidly and transiently released into circulation. Pretreatment with intravenous anti-TNF-alpha antibody reduced cortical (71%, P < 0.015) and subcortical (58%, P < 0.007) injury, enhanced the cerebral blood flow during reperfusion, and improved the neurologic outcome. This further supports the contention that TNF-alpha is a deleterious cytokine in stroke, whereas circulating antibody against TNF-alpha may protect brain from reperfusion injury.

Primary central nervous system lymphoma in homosexual men. Clinical, immunologic, and pathologic features.

Primary central nervous system lymphoma constitutes one of the criteria for the acquired immune deficiency syndrome (AIDS), yet a paucity of information is currently available regarding the clinical, immunologic, or pathologic features of these patients. Six homosexual men presenting with primary central nervous system lymphoma were evaluated. Five of these patients presented with altered mental status. All lymphomas were intracranial. B cell immunoblastic sarcoma was found in five. Immune phenotyping studies performed in five patients revealed monoclonal lambda light chain in three, whereas one expressed only IgG heavy chain, and one demonstrated another B cell (LN-1) surface antigen. Hypodense, contrast-enhancing lesions were apparent on computed axial tomographic scanning of the brain, in sharp contrast to isodense or hyperdense lesions reported in primary central nervous system lymphomas without underlying immunodeficiency. Immunologic abnormalities in these patients were similar to those in AIDS presenting as Kaposi's sarcoma or with opportunistic infections. In spite of therapeutic interventions, survival was short, and only one patient is currently alive.

HLA-DR (Ia)-positive dendritic-like cells in human fetal nonlymphoid tissues.

Nonlymphoid tissues from human fetuses ranging in age from 12 to 21 weeks' gestation were examined for the presence of HLA-DR (Ia)-positive cells. Immunoperoxidase staining of cryostat sections revealed Ia-positive cells with dendritic-like morphology, in kidney, heart, pancreas, and lung--but not in brain tissue. These Ia-bearing cells were present at 12 weeks' gestation, and by 21 weeks had increased to substantial numbers in all the organs tested, except for the brain, which remained negative. To further characterize the nature of these Ia-positive cells serial sections were studied with a panel of monoclonal antibodies (MAb) consisting of four reagents identifying monocyte/macrophages and two reagents staining lymphoid cells. The four anti-monocyte/macrophage MAb stained few if any cells in all the tissues examined. Lymphoid cells, as identified by anti-B-cell and anti-T-cell MAb, were also present in very low numbers. Additional studies using a double-staining technique provided direct evidence that the Ia-positive cells bear the human common leukocyte antigen detected by MAb T29 /33 and, with few exceptions, are negative for the macrophage antigen detected by MAb OKM1. The data suggest that nonlymphoid fetal tissues contain Ia-positive dendritic-like cells that are not monocyte/macrophage or lymphoid in origin. These Ia-bearing cells may be related to the dendritic cells found in lymphoid tissues, which are highly stimulatory in mixed leukocyte reactions and are thought to be responsible for the initiation of allograft rejection.

Extensive peroxynitrite activity during progressive stages of central nervous system inflammation.

Nitric oxide (NO) production has been associated with disease activity in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). This free radical can be transformed by superoxide to peroxynitrite, an extremely toxic oxidant which causes lipid peroxidation. In addition, peroxynitrite nitrates tyrosine residues, resulting in nitrotyrosine, which can be identified immunohistochemically. The results of this study indicate that peroxynitrite is formed very early during EAE development, correlating with clinical disease activity. Nitrotyrosine-positive cells display a widespread distribution in brain and spinal cord during severe disease and are associated with both perivascular infiltrates and parenchymal sites. Double-staining procedures demonstrated that a subpopulation of CD11b-positive cells (macrophages/microglia) reacted with nitrotyrosine antibodies. Immunostaining for inducible NO synthase demonstrated a similar distribution as nitrotyrosine staining. These experiments indicate that peroxynitrite is formed during progressive stages of disease activity.

Exogenous tat protein activates central nervous system-derived endothelial cells.

Tat protein, an HIV gene product known to be secreted extracellularly, was tested to determine its role in the dissemination of HIV into the central nervous system (CNS). Tat was shown to activate human CNS-derived endothelial cells (CNS-EC) by the increase in the expression of E-selectin, the synthesis of IL-6, and the secretion of plasminogen activator inhibitor-1 (PAI-1). Tat also functioned synergistically with tumor necrosis factor alpha (TNF). AIDS brains stained for tat in situ, demonstrated positive cells. These data suggest that secreted tat protein may increase leukocyte binding, and alter the blood-brain barrier permeability to enhance dissemination of HIV-infected cells into the CNS.

HIV-1 tat protein induces the production of interleukin-8 by human brain-derived endothelial cells.

This study focused on the role of the HIV-derived viral protein, tat, in activating central nervous system (CNS)-derived endothelial cells (EC) to produce interleukin-8 (IL-8), a stimulator and chemoattractant for neutrophils and lymphocytes. Human CNS-EC treated with tat (100 ng/ml) demonstrated a 2 to 3 fold upregulation in IL-8 mRNA and protein. Tumor necrosis factor-alpha (TNF) and tat were found to act additively in upregulating IL-8 production. In contrast, transforming growth factor beta (TGF beta), appeared to down modulate tat-induced IL-8 production. These data suggest that extracellular tat, especially in the presence of TNF, may be responsible for the local production of IL-8.

Endothelin-1 and monocyte chemoattractant protein-1 modulation in ischemia and human brain-derived endothelial cell cultures.

Brain tissue damage due to ischemia/reperfusion has been shown to be caused, in part, by activated macrophages infiltrating into the post-ischemic brain. Using the Middle Cerebral Artery Occlusion (MCAO) mouse model, this study demonstrated that, in vivo, both endothelin-1 (Et-1), a potent vasoconstrictor, and the macrophage chemokine, monocyte chemoattractant factor-1 (MCP-1) are induced in ischemia. Further studies, using human brain-derived endothelial cells (CNS-EC), showed that in vitro, Et-1 can directly stimulate MCP-1 mRNA expression and MCP-1 protein; and this Et-1-induced MCP-1 production is mediated by the ET(A) receptor. Inflammatory cytokines, tumor necrosis factor alpha and interleukin-1beta, functioned additively and synergistically, respectively, with Et-1 to increase this MCP-1 production. Partial elucidation of the signal transduction pathways involved in Et-1-induced MCP-1 production demonstrated that protein kinase C-, but not cAMP-dependent pathways are involved. These data demonstrate that Et-1, functioning as an inflammatory peptide, increased levels of MCP-1, suggesting a mechanism for chemokine regulation during ischemia/reperfusion injury.

TGF-B2 and soluble p55 TNFR modulate VCAM-1 expression in glioma cells and brain derived endothelial cells.

Transforming growth factor beta-2 (TGF-B2) is secreted by glioma cells and is known to decrease leukocyte-endothelium interaction. TGF-B2 alone and in conjunction with soluble tumor necrosis factor (TNF) p55 receptor, was found to decrease the expression of TNF induced VCAM-1 on the malignant glioma cell line A-172 and human cerebral microvessel endothelial (CNS-EC) cells. Co-culture of A-172 glioma cells led to a decrease in VCAM-1 expression; this effect on CNS-EC in co-culture could be simulated by glioma supernatant alone. These results suggest that malignant gliomas, by secreting TGF-B2 and releasing soluble TNF receptors, modulate adhesion molecules.

Tumor necrosis factor-alpha in the retina in acquired immune deficiency syndrome.

The presence of specific cytokines and the number and distribution of leukocytes were determined in the retinas of patients with acquired immune deficiency syndrome (AIDS). Using immunohistocytochemical techniques, three retinas from patients with AIDS had focal infiltration by T-lymphocytes and macrophages. These specimens stained positively for tumor necrosis factor-alpha (TNF-alpha) in cells identified morphologically as macrophages and glial cells and showed prominent reactive gliosis. The retinas from seven other affected patients had minimal leukocytic infiltration and no TNF-alpha reactivity; gliosis was present in only one. The retinas from clinically normal patients without human immunodeficiency virus (HIV) contained no TNF-alpha-positive cells. Using in situ hybridization for HIV, four of five patients with AIDS had rare positive cells. No interferon-gamma was detected in any of the retinal tissues tested. These data suggest a role for TNF-alpha in the development of AIDS-related retinal disease.

Class II antigen expression by lacrimal epithelial cells. An updated working hypothesis for antigen presentation by epithelial cells.

It has been suggested that aberrant expression of Class II histocompatibility antigens (HLA) is involved in T cell activation and leads to autoimmunity. Although Class II antigen expression was found in various nonlymphoid tissues, including salivary glands, its expression on lacrimal epithelial cells has not been reported. In this study, 12 cadaver lacrimal glands were analyzed for HLA-DR and for the numbers and distributions of T suppressor cells (Ts), T helper cells (Th), B cells, and macrophages. None of these cases exhibited the high numbers of inflammatory cells, tissue damage, and fibrosis characteristic of Sjogren's syndrome. The HLA-DR-positive epithelial cells were detected in ten cases; they represented from less than 1% to more than 70% of the epithelial cells. In these ten positive cases, there were greater numbers of T cells per millimeter squared (229 +/- 94 [mean +/- the standard error of the mean]) than in the two HLA-DR-negative cases (37 +/- 1 [mean +/- range]). Three lacrimal gland specimens tested were negative for immunoglobulin (Ig) G-bearing B cells, and two of the three specimens tested had IgA-bearing cells. Acinar cells were isolated from rat and rabbit lacrimal glands and cultured overnight in serum-free media supplemented with several potential mediators of Class II antigen expression: interferon-gamma, carbachol, or isoproterenol. Freshly isolated cells did not express Class II antigens at detectable levels, but in most experiments, they began to express the antigen even in the absence of putative mediators.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of intercellular adhesion molecule-1 by tumor necrosis factor-alpha through the 55-kDa receptor is dependent on protein kinase C in human retinal pigment epithelial cells.

PURPOSE: To determine second messenger signaling pathways associated with tumor necrosis factor-alpha (TNF)-mediated induction of intercellular adhesion molecule (ICAM)-1 expression on human retinal pigment epithelial (HRPE) cells, a cell type known to express only the 55-kDa TNF receptor (TNFR p55). METHODS: SV 40-immortalized HRPE (SVRPE) cells were exposed to TNF with and without pretreatment with the protein kinase C (PKC) inhibitor calphostin C or the protein kinase A (PKA) inhibitor H8. SV40-immortalized HRPE cells also were treated with the PKC activator phorbol 12-myristate 13-acetate (PMA) or with the PKA activators forskolin plus 3-isobutyl-1-methyl-xanthine or dibutyryl cyclic adenosine monophosphate (cAMP) alone. Membrane fractions from untreated and treated SVRPE cells were assayed for PKC activity, and whole cell lysates were assayed for cAMP accumulation and PKA activity. Flow cytometry was performed on SVRPE cells using a monoclonal antibody specific to ICAM-1. RESULTS: Activation of TNFR p55 on SVRPE cells with TNF resulted in a rapid increase of PKC activity at 1 minute, with a subsequent downregulation to baseline. There was no increase in intracellular cAMP accumulation or PKA activity within the first 10 minutes; however, both increased within 30 minutes and returned to baseline within 1 hour. SV40-immortalized HRPE cells treated with TNF for 1 hour showed maximal induction of ICAM-1 expression at 18 hours. ICAM-1 induction by TNF treatment was inhibited by calphostin C pretreatment and not by H8 pretreatment. Protein kinase C activation with PMA for 3 hours was sufficient to induce ICAM-1 on SVRPE cells at 18 hours, whereas treatment with the PKA activators forskolin or dibutyryl cAMP did not induce ICAM-1 expression. CONCLUSIONS: Tumor necrosis factor sequentially activates the PKC and PKA pathways in SVRPE cells by way of the TNFR p55. The PKC pathway in necessary for TNF-mediated ICAM-1 upregulation, and specific activation of the PKC pathway with PMA is sufficient to induce ICAM-1 on these cells. SV40-immortalized HRPE cells may serve as a model in which to study further the functional signaling pathways associated with TNFR p55.

Stability of T- and B-cell numbers in human peripheral blood.

A comparison was made between the percentage of T and B cells present in human blood on the day of collection and those recovered from whole blood specimens stored for one, two, and three days. In the peripheral blood of normal individuals, the percentage of E-rosetting and surface-membrane immunoglobulin-positive cells was unchanged throughout the three day period. Furthermore, whole blood samples from patients with various hematological diseases maintained for three days exhibited T- and B-cell percentages equivalent to those tested on day zero.

Immunohistology of persistent generalized lymphadenopathy. Evidence for progressive lymph node abnormalities in some patients.

Immunohistologic analysis of cellular changes in serial lymph node biopsies of eight patients with persistent generalized lymphadenopathy (PGL) syndrome was performed and correlated with clinical and laboratory findings to better determine the natural history of human immunodeficiency virus (HIV) infection. The authors observed decreased follicle size and area in the second biopsies of six of the eight patients, associated in some with increased numbers of B-cells in medullary regions (four of eight) and more involuted follicles (four of eight). Five cases showed progressively increased paracortical areas in the second biopsies, with increased numbers of T-cytotoxic/suppressor cells and decreased T-helper cells. Seven of the patients also had a progressive loss of T-helper cells in the peripheral blood. These findings provide tissue and peripheral blood evidence for progressive immunologic deterioration in some patients with PGL.

Regional distribution of thrombomodulin in human brain.

Thrombomodulin, an integral membrane protein with important anticoagulant properties, was evaluated for its distribution in blood vessels of human brain. Adjacent sections from frozen human brain were stained for von Willebrand Factor (an endothelium marker) or thrombomodulin. The number of thrombomodulin-positive blood vessels was expressed as a percentage of all blood vessels in a specific area. The density ratio was compared among cortical and subcortical regions in the human brain. Our results indicated that thrombomodulin density in neocortex, cerebellum, medulla and hippocampus was similar. There was, however, significantly less thrombomodulin in putamen (P less than 0.01), pons (P less than 0.001) and mesencephalon (P less than 0.001) compared to neocortex. The regional distribution of thrombomodulin may prove to be helpful for understanding the development of thrombotic diseases of the brain.

Differential effects of tumor necrosis factor-alpha on proliferation, cell surface antigen expression, and cytokine interactions in malignant gliomas.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine produced by astrocytes in vivo and in vitro, was tested for its effects on two malignant astrocytoma cell lines (A-172, U-87). Both lines were immunoreactive for glial fibrillary acidic protein, vimentin, Class I antigens, and interleukin-6. The lines differed in their expression of Class II and intercellular adhesion molecule-1 (ICAM-1) antigenic determinants: A-172 cells were negative for both Class II and ICAM-1 antigens, while U-87 cells were intensely positive for Class II and weakly positive for ICAM-1. When these astrocytoma cell lines were exposed to TNF-alpha, A-172 growth was stimulated while U-87 growth was inhibited. Furthermore, in U-87 cells, TNF-alpha enhanced both ICAM-1 and interleukin-1 beta (IL-1 beta) expression, and decreased immunoreactivity for transforming growth factor-beta (TGF-beta) protein. In contrast, in the presence of TNF-alpha, A-172 cells remained negative for IL-1 beta and TGF-beta, but showed an increased expression of ICAM-1. These results demonstrate that TNF-alpha can induce changes in growth rate, cytokine production, and surface antigen expression in malignant astrocytomas; however, the nature of these changes is dependent on the specific characteristics of these malignant astrocytomas. The resultant variability in the immunological microenvironment of these tumors may reflect differences in their growth potential.

Cytokine expression in radiation-induced delayed cerebral injury.

Radiation-induced delayed brain injury is a well-documented complication of both standard external beam radiation (teletherapy) and interstitial brachytherapy; however, the cause of this damage has not been determined. Cytokines and growth factors are important regulatory proteins controlling the growth and differentiation of normal and malignant glial cells, which have been implicated in the tissue response to radiation injury. Six snap-frozen brain biopsies showing radiation injury were obtained from four patients harboring malignant gliomas who underwent either postoperative external beam and/or stereotactic interstitial brachytherapy at standard dosages. The specimens showed variable amounts of gliosis, tissue necrosis, calcification, inflammation, and vascular proliferation and hyalinization. Frozen tissue sections were examined for the presence of infiltrating lymphocytes, macrophages, cytokines, and other immunoregulatory molecules by the use of a panel of specific monoclonal and polyclonal antibodies. All specimens showed diffuse T cell infiltration with both CD4+ and CD8+ cells. Infiltrating activated macrophages (CD11c+, HLA-DR+) were prominent in five of six cases. Tumor necrosis factor-alpha and interleukin-6 immunoreactivity was prominent in four of six cases and was predominately localized to macrophages. Transforming growth factor-beta astrocytic and macrophage immunoreactivity was present at moderate levels in all cases. This study suggests that in radiation necrosis, interleukin-1 alpha, tumor necrosis factor-alpha, and interleukin-6 are expressed, predominately by infiltrating macrophages.