Michael N. Hart

CNS antigen presentation

Presentation of antigens for the CNS follows the same general rules as for other tissues. However, the presence of special CNS cells with immune functions plus the blood-brain barrier (BBB) suggests that differences in the way that the immune system functions in the CNS might help to explain why some autoimmune diseases are unique to the CNS. Irrespective of whether CNS antigen presentation takes place inside or outside the CNS (or both), the BBB clearly plays a major role in CNS immune function. The BBB governs the quantity and type of lymphocytes that enter the CNS by way of specific adhesion-molecule binding between lymphocytes and endothelium and possibly by selecting for antigen-specific lymphocytes in antigen-recognition events.

Activated/effector CD4+ T cells exacerbate acute damage in the central nervous system following traumatic injury

CD4(+) helper T cells (Th) have been demonstrated to participate in the chronic phase of traumatic injury repair in the central nervous system (CNS). Here, we show that CD4(+) T cells can also contribute to the severity of the acute phase of CNS traumatic injury. We compared the area of tissue damage and the level of cellular apoptosis in aseptic cerebral injury (ACI) sites of C57BL/6 wild type and RAG1(-/-) immunodeficient mice. We demonstrate that ACI is attenuated in RAG1(-/-) mice compared to C57BL/6 animals. Adoptive transfer of CD4(+)CD62L(low)CD44(high) activated/effector T cells 24 h prior to ACI into RAG1(-/-) mice resulted in a significantly enhanced acute ACI that was comparable to ACI in the C57BL/6 animals. Adoptive transfer of CD4(+)CD62L(high)CD44(low) naive/non-activated T cells did not increase ACI in the brains of RAG1(-/-) mice. T cell inhibitory agents, cyclosporin A (CsA) and FK506, significantly decreased ACI-induced acute damage in C57BL/6 mice. These results suggest a previously undescribed role for activated/effector CD4(+) T cells in exacerbating ACI-induced acute damage in the CNS and raise a novel possibility for acute treatment of sterile traumatic brain injury.

Influence of adhesion molecule expression by human brain microvessel endothelium on cancer cell adhesion.

Cultures of endothelial (En) cells derived from human brain microvessels were established in order to characterize adhesion molecule expression and to assay the adhesion properties of neoplastic cell lines to monolayers of En cells. Low constitutive expression of beta1 integrin (CD29), and ICAM-2 (CD102) was detected on human brain microvessel En cells. The beta1 chain of the VLA integrin family, ICAM-1, E-selectin (CD62E) and VCAM-1 (CD106) but not ICAM-2 and PECAM-1 (CD31) expression was upregulated by IL1-alpha, and TNF-alpha proinflammatory cytokines. High expression of PECAM-1 was found on non-activated human brain EN cells. In order to study the potential role of adhesion molecules in neoplastic cell adhesion two tumor cell lines were chosen. Adhesion of a cell line (DU145) derived from a cerebral metastasis of prostate carcinoma to human brain microvessel En cell monolayers was less pronounced compared to adhesion of a primary prostate carcinoma cell line (ND1). Adhesion of cerebral metastatic neoplastic cell line (DU145) was not significantly influenced by incubation of endothelial cells with different proinflammatory cytokines. The adhesion capability of primary prostate carcinoma line (NDI) was significantly upregulated by TNF-alpha proinflammatory cytokine. Furthermore, the adhesion of ND1 was partly inhibited using anti-E-selectin and VCAM-1 monoclonal antibodies. There was no significant effect of anti-adhesion antibodies on the adhesion characteristics of the cerebral metastatic (DU145) cell line. Our data demonstrate that different mechanisms are involved in the adhesion of neoplastic cells to cerebral En cells and turn our attention to the importance of adhesion molecule expression in the formation of metastases.

Traumatic brain injury increases TGFβRII expression on endothelial cells

Transforming growth factor beta (TGFβ) modulates a variety of growth related functions following traumatic injury. The cellular response to TGFβ is predominantly mediated through TGFβ receptor I (TGFβRI) and receptor II (TGFβRII) on the cell surface and SMAD proteins intracellularly. We investigated the expression of TGFβ receptors in the acute and chronic phases of a traumatic cerebral injury (TCI) by immunohistochemistry and in cultures of murine brain microvascular endothelial (EN) cells using cytofluorimetry. Here, we report that TGFβRII expression significantly increases on brain endothelial cells in the chronic phase of TCI. SMAD3 and SMAD4 protein expression were also upregulated suggesting the activation of TGFβ receptor intracellular signaling. When TGFβRI and TGFβRII expression was studied in in vitro cultures of murine brain microvessel EN cells, TGFβRII showed increased expression on proliferating cells that are incorporating BrdU. These data show a differential expression of TGFβRI and TGFβRII on brain microvessel EN cells in the acute and chronic phases of TCI that might be associated with EN proliferation following injury.

Autoantibodies to vascular smooth muscle are pathogenic for vasculitis.

We have previously shown that microvascular smooth muscle activates CD4+ T lymphocytes in sterile co-culture, presents antigen, and produces inflammatory cytokines. Adoptive transfer of lymphocytes co-cultured with syngeneic smooth muscle cells to healthy recipient mice results in vasculitic lesions predominantly in postcapillary venules. The present study assessed the pathogenic role of immunoglobulin and B cells in a murine model of vasculitis. Here, we show that transferred B cells, including plasmablast cells, accumulated, persisted, and proliferated in lung and secondary lymphoid organs of recipient mice. The induction of vasculitis was accompanied by production of IgM and IgG2a autoantibodies specific for vascular smooth muscle intracellular antigens. Circulating immunoglobulin had a pathogenic role in this vasculitis model, because the disease could be induced by transfer of serum from vasculitic mice to untreated animals but not by transfer of serum depleted of anti-smooth muscle autoantibodies. Additionally, the pathogenic mechanisms triggered by the transfer of vasculitogenic serum were dependent on T lymphocytes because both wild-type and B cell-deficient mice developed the disease after serum transfer, whereas RAG2-deficient mice did not. Thus, immunoglobulin and cell-mediated pathways work in concert to produce vasculitis in this model.

A small population of vasculitogenic T cells expands and has skewed T cell receptor usage after culture with syngeneic smooth muscle cells

Adoptive transfer of lymphocytes co-cultured with syngeneic smooth muscle (SM) cells to healthy recipient mice results in vasculitic lesions predominantly in post-capillary venules. The present study focuses on the mechanisms by which the disease-inducing CD4(+) T cells are generated in co-culture of lymphocytes with SM cells. Microvascular SM cells provide survival signals to both CD4(+) and CD8(+) naïve syngeneic T cells and can activate only a limited range of CD4(+) T lymphocytes in culture. Additionally, approximately 0.4% of the original CD4(+) T cells divide at least twice in co-culture with SM cells. Survival of CD4(+) T cells in co-culture is dependent on a TCR mediated process, since transgenic CD4 (+)cells with a unique specificity for a non-murine peptide do not survive in culture with SM. Analysis of TCR Vbeta shows no superantigen activation of T cells following co-culture with SM cells. Spectratype analysis of TCR Vbeta Jbeta segment usage reveals a skewage in the TCR repertoire of T cells co-cultured with SM, and also of T cells from vasculitic lung. These results are consistent with a specific immune response of pathogenic T cells against one or more activating antigenic determinants of the microvascular SM cells, in contrast to non-specific cytokine activation.

Amyloid-β Deposition in Skeletal Muscle of Transgenic Mice

Inclusion body myopathy is a progressive muscle disorder characterized by nuclear and cytoplasmic inclusions and vacuolation of muscle fibers. Affected muscle fibers contain deposits of congophilic amyloid, amyloid-beta immunoreactive filaments, and paired helical filaments, all of which are pathological hallmarks of Alzheimers disease in brain. Accumulations of amyloid-beta and its precursor are thought to play important roles in the pathogenesis of both inclusion body myopathy and Alzheimers disease. Overexpression of mutant forms of beta protein precursor in transgenic mice by neuron-specific promoters has been reported to cause amyloid deposits in the brain. Here we report that overexpression in transgenic mice of the signal plus 99-amino acid carboxyl-terminal sequences of beta protein precursor, under the control of a cytomegalovirus enhancer/beta-actin promoter, resulted in vacuolation and increasing accumulation of the 4-kd amyloid-beta and the carboxyl-terminus in skeletal muscle fibers during aging. These deposits in transgenic muscle only rarely showed Congo red birefringence. Thus, overexpression of part of beta protein precursor in transgenic mice led to development of some of the characteristic features of inclusion body myopathy. These mice may be a useful model of inclusion body myopathy, which shares a number of pathological markers with Alzheimers disease.

CD4+ T Cells Sensitized by Vascular Smooth Muscle Induce Vasculitis, and Interferon Gamma Is Critical for the Initiation of Vascular Pathology

Primary vasculitis is the result of idiopathic inflammation in blood vessel walls. T cells are believed to play a critical role, but the nature of the pathological T-cell response remains obscure. In this study, we provide evidence that CD4(+) T lymphocytes, activated in the presence of syngeneic vascular smooth muscle cells, were sufficient to induce vasculitic lesions after adoptive transfer to recipient mice. Additionally, the disease is triggered in the absence of antibodies in experiments in which both the donors of stimulated lymphocytes and the transfer recipients were mice that were deficient in B cells. Tracking and proliferation of the transferred cells and their cytokine profiles were assessed by fluorescence tagging and flow cytometry. Proliferating CD4(+) T cells were evident 3 days after transfer, corresponding to the occurrence of vasculitic lesions in mouse lungs. The transferred T lymphocytes exhibited Th1 and Th17 cytokine profiles and minimal Th2. However, 1 week after vasculitis induction, effector functions could be successfully recalled in Th1 cells, but not in Th17 cells. Additionally, in the absence of constitutive interferon-γ expression, T cells sensitized by vascular smooth muscle cells failed to induce vasculitis. In conclusion, our results show that Th1 cells play a key role in eliciting vasculitis in this murine model and that induction of the disease is possible in the absence of pathogenic antibodies.

Murine Endothelia Do not Express MHC Class II I-Eα Subunit and Differentially Regulate I-Aα Expression along the Vascular Tree

Cellular elements of the vascular wall, such as endothelium (En) and smooth muscle cells/pericytes (SM/P) possess important immunologic properties. We have previously reported that murine brain microvessel En cells and SM/P express Major Histocompatibility (MHC) class II molecules and activate syngeneic CD4+ T cells in a class II dependent way. Herein we compare MHC class II expression on brain microvessel En to aorta large vessel En cells in order to explore the mechanisms of immune responses in brain tissue versus other peripheral tissues. Interestingly, we demonstrate that En cells from brain microvessel and large aortic vessel express the I-A but not the I-E subunit of MHC class II molecules. The expression of I-A class II molecules can be upregulated on brain microvessel and aortic En cells by interferon-γ (IFN-γ). Similarly, the expression of I-A, but not I-E, MHC class II molecules on brain microvessel endothelial cells was upregulated in the presence of activated T cells. Interleukin-10 (IL-10) wa...

The role of antigen specificity in induction of T cell accumulation in the CNS

Based on the observation that the application of monoclonal antibodies (mAbs) directed against ct4-integrin and VCAM-I inhibit the development of experimental autoimmune encephalomyelitis (EAE) in vivo it has been concluded that the successful therapeutic effect is due to interference of the mAbs with aal]I/VCAM-I mediated interaction of autoaggressive T ceils with the blood-brain barrier (BBB). However. a possible role of ~x4[~7-integrin. which also binds to VCAM-I, or interference with other T cell mediated events during the pathogenesis of EAE has not been considered. We have compared the effects of mAb therapy on the development of EAE in the SJL/J mouse, using a large panel of mAbs directed against the ct4-subunit, the l~l-subunit, the !~7-subunit, the a4ta7-integrin and against VCAM-I, Although encephalitogenic T cells express both ct4-integrins, mAbs directed against ct,q~7 or the flT-suhunit did not intertere with the development of EAE. In contrast, mAbs directed against a4 and VCAM-I clearly inhibited or diminished clinical and histopathological signs of EAE. Therefore, ct41~l but not ct4137 is critically involved in the development of EAE. Furthermore, our in vitro studies suggest that the therapeutic effect of anti-a4-treatment of EAE might not merely be due to inhibition of asI~I/VCAM-I mediated T cell interaction with the BBB. The possible influence of anti-ct4-treatment on T cell function in EAE will be discussed. 98 The Anaphylatoxin Receptor for Complement C3a is Expressed by Rat Mieroglia and Rat Oligodendrocytes. Role in Cell Chemotaxis and Cell Activation. P. Gagque, S.K. Singhrao, J.W. Neal , UWCM, U.K., J.D. Sedgwick, Centenary Institute of Cancer Medicine & Cell Biolo~, Australia