Biomedical subjects
Publications and source records attributed to D Male.
The genomic matching technique has proven useful in MHC haplotyping in humans. We have adopted a similar approach in Australian cattle dogs and report that genotyping can be achieved with a single assay.
The selective accumulation of different leucocyte populations during inflammation is regulated by adhesion molecules and chemokines expressed by vascular endothelium. This study examined how chemokine production and the expression of adhesion molecules and chemokine receptors vary between endothelia from different vascular beds. Human saphenous vein endothelium was compared with lung and dermal microvascular endothelia and with umbilical vein endothelium and a bone-marrow endothelial cell line. All endothelia produced CCL2 and CXCL8 constitutively, whereas CXCL10 and CCL5 were only secreted after tumour necrosis factor (TNF)-alpha or interferon (IFN)-gamma stimulation. In combination with TNF-alpha, IFN-gamma suppressed CXCL8 but enhanced CCL5 and CXCL10, whereas transforming growth factor (TGF)-beta reduced secretion of all chemokines. Basal chemokine secretion was higher from umbilical vein than other endothelial cells. Chemokine receptors, CXCR1, CXCR3 and CCR3, were present on all endothelia but highest on saphenous vein. CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 were also detected at variable levels on different endothelia. The variation between endothelia in chemokine secretion was much greater than the variations in adhesion molecules, both on resting cells and following cytokine stimulation. These results indicate that it is the tissue-specific variations in endothelial chemokine secretion rather than variations in adhesion molecules that can explain the different patterns of inflammation and leucocyte traffic seen in non-lymphoid tissues.
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We conducted a double-blind randomised controlled trial comparing the efficacy of analgesia during labour of remifentanil and pethidine. Nine women were randomised to receive an i.v. bolus of remifentanil 0.5 microg.kg(-1)with a lockout period of 2 min and eight women were randomised to receive a bolus of pethidine 10 mg with a lockout period of 5 min. A visual analogue scale (VAS) scoring system was used to assess the level of pain hourly throughout the first and second stages of labour and a score was recorded within half an hour of delivery for the level of pain overall throughout labour (post delivery score). The study was terminated after 17 subjects, on agreement with the local ethics committee, due to concern with the poor Apgar scores in the pethidine group. With the data available, we demonstrated significantly lower mean hourly and post delivery VAS scores for pain in the remifentanil group (P < 0.05). The 1 and 5 min Apgar scores were significantly lower in the pethidine group compared with the remifentanil group (P < 0.05). This preliminary study suggests that remifentanil may have a use as patient-controlled analgesia for women in labour.
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterised by perivascular inflammatory cell infiltrates and plaques of demyelination. Chemokines have been shown to play an important role in the activation and directional migration of cells to sites of CNS inflammation. The action of chemokines requires the expression of their complementary chemokine receptors by their target cells. We have examined the expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in post-mortem MS CNS tissue using single- and double-labelling immunocytochemistry techniques. Low levels of CCR2, CCR3 and CCR5 were expressed by microglial cells throughout control CNS tissue. In chronic active MS lesions CCR2, CCR3 and CCR5 were associated with foamy macrophages and activated microglia. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes. A smaller number of CCR3-positive lymphocytes were present, but we also noted CCR3 and CCR5 on astrocytes in five of the 14 cases of MS investigated, particularly associated with processes around vessels and at the glia limitans. Ligands for CCR2 and CCR3 include MCP-1 and MCP-3 which were co-localised around vessels with the infiltrating leukocytes, but were also present in unaffected areas of cortex. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS.
Experimental allergic encephalomyelitis (EAE) is a frequently employed animal model of the human disease multiple sclerosis. EAE can be induced by adoptive transfer of CD4+ T cells that are specific for central nervous system (CNS) antigens, typically myelin proteins. Although the pathogenic mechanism or mechanisms responsible for the clinical signs and histological changes in EAE and multiple sclerosis are not fully defined, the entry of T lymphocytes and antigen recognition within the CNS are required. The present study describes the participation of a novel cell surface molecule with properties suggesting a role in cell-cell adhesion or co-stimulation, or both, in the development of EAE in the rat. The molecule is defined by the unique monoclonal antibody (mAb) TLD-4A2. The TLD-4A2 antigen is present on resting and activated T lymphocytes, activated CNS endothelial cells, and microglia. The antigen is normally distributed in many tissues including lymph node, thymus, and spleen, as well as in the inflamed CNS. Both its pattern of tissue distribution and immunoprecipitation and immunoblotting studies suggest that the TLD-4A2 antigen is a novel molecule. Treatment of rats with the purified 4A2 mAb resulted in the inhibition of the clinical signs of EAE and also decreased the number T cells and macrophages accumulating in the CNS parenchyma. TLD-4A2 antibody did not seem to directly interfere with T cell viability in vivo, as demonstrated by the ability to recover and stimulate CD4+ encephalitogenic T cells from cervical lymph nodes of 4A2-treated animals. In vitro, the antibody partially blocked T cell proliferation assays. These data suggest that the TLD-4A2 mAb recognizes a novel molecule expressed on lymphocytes, endothelial cells, and macrophages that may play a role in hematogenous cell traffic and the initiation of CNS inflammation.
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Neuropathologically, human immunodeficiency virus (HIV) is associated with a range of inflammatory disorders, extensive cortical neuronal loss, and dendritic and synaptic damage. Although the mechanisms resulting in these abnormalities are still unclear, the neurotoxic effects are thought to be due in part to viral products including the tat gene product. We have previously shown that Tat when presented to neurons extracellularly interacts with neuronal cell membranes to cause neuronal excitation and toxicity in fmole amounts. To determine the role of Tat in mediating HIV encephalitis (HIVE), we detected tat mRNA and protein in tissue extracts of nine patients with HIVE and seven patients without HIVE. Despite long autopsy times and significant degradation, tat mRNA was detected in 4/9 patients with HIVE but not in any of the seven patients without dementia. Similarly, the env mRNA was also detected in 5/9 patients with HIVE but not in the patients without HIVE. However, vif mRNA was detected in both groups of patients with (5/9) or without (2/7) HIVE. Using protein extracts from the brains of the same groups of patients we were unable to detect Tat by enzyme linked immunosorbant assay (ELISA) (sensitivity of 2 ng Tat/ml of brain tissue). However, Tat could be detected immunohistochemically and in protein extracts from the brains of rhesus macaques with encephalitis due to a chimeric strain of HIV and simian immunodeficiency virus (SHIV). Our observations support the role of Tat in the neuropathogenesis of HIV and SHIV encephalitis.
Microglia are the immune effector cells of the nervous system. The prevailing view is that microglia are derived from circulating precursors in the blood, which originate from the bone-marrow. Colonisation of the central nervous system (CNS) by microglia is an orchestrated response during human fetal development related to the maturation of the nervous system. It coincides with vascularisation, formation of radial glia, neuronal migration and myelination primarily in the 4th-5th months and beyond. Microglial influx generally conforms to a route following white matter tracts to gray areas. We have observed that colonisation of the spinal cord begins around 9 weeks, with the major influx and distribution of microglia commencing around 16 weeks. In the cerebrum, colonisation is in progress during the second trimester, and ramified microglial forms are widely distributed within the intermediate zone by the first half of intra-uterine life (20-22 weeks). A distinct pattern of migration occurs along radial glia, white matter tracts and vasculature. The distribution of these cells is likely to be co-ordinated by spatially and temporally regulated, anatomical expression of chemokines including RANTES and MCP-1 in the cortex; by ICAM-2 and PECAM on radiating cerebral vessels and on capillaries within the germinal layer, and apoptotic cell death overlying this region. The phenotype and functional characteristics of fetal microglia are also outlined in this review. The need for specific cellular interactions and targeting is greater within the central nervous system than in other tissues. In this respect, microglia may additionally contribute towards CNS histogenesis.
The migration of lymphocytes through primary cultures of rat brain microvascular endothelial cell monolayers was examined in vitro by time-lapse videomicroscopy. Antigen-specific T cell line migration was dependent on the duration of culture (post-antigen stimulation) with exogenous interleukin-2 (IL-2). Peak migration (approximately 50% of T-cells during the 4 h migration assay) occurred after 4 days of culture with IL-2 but did not coincide with maximal expression of LFA-1, VLA-4 or the IL-2 receptor. On unstimulated endothelia antibody blockade of LFA-1 or ICAM-1 inhibited T-cell line migration to 8.0% and 6.8% of control values, respectively, whereas blocking VLA-4 and VCAM-1 had no effect. On IL-beta activated endothelium blocking LFA-1 and ICAM-1 was less effective (24.9% and 27.3% of control values, respectively) and blockade of VLA-4 and VCAM-1 brought about a reduction to 63.0% and 68.3% of controls respectively. Inhibition of IL-2-dependent proliferation with an IL-2 receptor blocking antibody also significantly inhibited T-cell migration to 22.2% of controls. Peripheral lymph node (PLN) lymphocytes could also be induced to migrate through untreated cerebral endothelial cell monolayers by cross-linking CD3 which was also time and IL-2-dependent with maximal migration (22.7%) occurring after three days in the presence of exogenous IL-2. Blocking LFA-or ICAM-1 resulted in a significant reduction in migration across IL-1 beta-activated endothelial cells to 17.4% and 20.9% of control values respectively although blocking the VLA-4/VCAM-1 interaction had no significant effect. Activation of PLN lymphocytes with concanavalin A for up to 5 days did not induce migration but when left in contact with the endothelial monolayer for 24 h migration reached 31.0%. These studies indicate that T-cells require a combination of signals to trigger the migratory phenotype which is necessary to enable them to penetrate the blood-brain barrier.
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We undertook a search for cytokine-inducible molecules present on brain endothelium and which are involved in the control of lymphocyte adhesion. We screened 39 monoclonal antibodies (mAb) against rat brain endothelium in vitro, and identified five recognizing cytokine-inducible molecules. None of the 39 antibodies blocked lymphocyte adhesion, but one antibody (4A2), produced a 400% enhancement of lymphocyte binding. The 4A2 antigen is induced on brain endothelium by interferon-gamma (INF-gamma) but not tumour necrosis factor-alpha (TNF-alpha), at 6-48 hr. It is preferentially expressed near inter-endothelial cell junctions, but it also expressed on all lymphocytes and weakly on aortic endothelium in vitro. In vivo, it is not detectable on cells in the normal central nervous system (CNS), however it appears in the CNS during T-cell mediated immune reactions. Triggering of cells via this molecule enhances integrin-mediated adhesion of lymphocytes to brain endothelium, primarily via LFA-1. Unlike ICAM-1, 4A2 antigen is induced on endothelium of both Lewis and PVG strains. Although, it has some functional properties of human CD31, the 4A2 antigen is not rat CD31. The cellular localization of this molecule, its actions on integrin-mediated adhesion and its induction by IFN-gamma, all indicate that the 4A2 antibody recognizes a molecule involved in the control of lymphocyte migration into the brain.
We describe a recently developed assay for the analysis of leukocyte migration across cerebral endothelium in vitro. The endothelium is grown as monolayers on Goretex or Cyclopore membranes coated with extracellular matrix proteins and supported on inserts. This system permits the recovery and phenotyping of cells which migrate down through the endothelium. Using labelled lymphocytes we were able to differentiate four populations of cells, with differing degrees of mobility in the migration assay. We have compared the results from this system with those from conventional adhesion assays. Binding of cells to the endothelium is rapid, but is confined to a particular subpopulation of the applied lymphocytes. We have followed cell migration over 24 h in the system using normal and cytokine-activated endothelium and have found that whereas adhesion depends both on the state of lymphocyte activation and on the condition of the endothelium, the level of migration of stimulated lymphocytes is largely independent of endothelial activation. Moreover, whereas CD8+ cells bind well to the endothelium, it is the CD4+ cells which migrate most effectively. Comparison of brain and epididymal fat endothelium showed similar migration levels over 2 h, but migration was greater across epididymal fat endothelium at 24 h.
We examined the changes in intercellular adhesion molecule-1 (ICAM-1) expression on brain endothelium in response to tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). ICAM-1 is normally present on these cells and is induced over 24 hr by both cytokines with a time-course which matches enhancement in lymphocyte adhesion. Anti-lymphocyte function-associated antigen-1 (anti-LFA-1) (CD11a), anti-very late antigen-4 (anti-VLA-4) (CD49d) and anti-CD18 block binding of mitogen-activated lymphocytes to brain endothelium and the effects of anti-LFA-1 and anti-VLA-4 are additive. Anti-ICAM-1 does not however block adhesion, nor does depletion of endothelial ICAM-1 reduce lymphocyte binding. Titration of the interacting cells indicated that the antibody blocking is due to interference in the endothelial/lymphocyte interaction. None of the antibodies affect the binding of non-activated lymphocytes, which is itself normally much lower than that of activated cells. The time at which lymphocyte adhesiveness is greatest for the endothelium corresponds with the time at which the lymphocytes express highest levels of LFA-1 and VLA-4. The data show a role for LFA-1 and VLA-4 in the early interaction of activated lymphocytes with brain endothelium. Kinetic studies indicate that the ligand for VLA-4 is VCAM-1. The ligand for LFA-1 could not be determined with certainty, but if it is ICAM-1, the levels of ICAM-1 on brain endothelium are not critical.
We report on a series of experiments which examines the factors controlling lymphocyte adhesion to brain endothelium in vitro and the factors which control cell migration across the endothelium, using a new migration assay. Although lymphocyte adhesion preceded migration across the brain endothelium, the two processes are not identical. We noted that activated CD4+ T cells were particularly good at migrating across endothelia. CD8+ T cells and B cells did not migrate but adhered well to endothelia. Moreover, the endothelium maintained high levels of cell traffic without being disrupted and without exhausting the molecular systems which allowed migration. From the viewpoint of migration of dividing cells, the state of lymphocyte activation appeared to be the most important controlling factor--these cells migrated equally well across endothelium activated with cytokines or untreated endothelium. The kinetics of adhesion suggested that the LFA-1/ICAM-1 and VLA-4/VCAM combinations of adhesion molecules were important in controlling migration. With antibody blocking studies, the role of the LFA-1/ICAM-1 system was equivocal. While anti-LFA-1 blocked lymphocyte adhesion, anti-ICAM-1 did not, suggesting that the level of ICAM-1 was not critical.
Factors controlling lymphocyte adhesion to brain endothelium were investigated in vitro. Mitogen activation of lymphocytes causes increased adhesion to endothelium, which is maximal at 7-24 hr, declines to normal levels after the cells divide, and requires protein synthesis. Rat brain endothelium can be stimulated with IFN-gamma to increase its adhesion to either normal or activated lymphocytes. The endothelium is sensitive to low levels of cytokine: adhesion develops rapidly after stimulation and requires new protein synthesis. Antigen-specific line cells also adhere more effectively to endothelium than normal lymph node cells. This is enhanced by IFN-gamma treatment of the endothelium and is further increased marginally in the presence of the cognate antigen. The results suggest that either local stimulation of endothelium with cytokines or lymphocyte activation in the periphery will modulate lymphocyte traffic into brain.
The immunological properties of cerebral microvascular endothelium were directly compared with those of an extra-cerebral endothelium in vitro. Lymphocyte adhesion to cerebral endothelium is normally low, but is sensitive to induction by interferon-gamma (IFN gamma) and tumour necrosis factor-alpha (TNF alpha). Conversely adhesion to aortic endothelium is normally much higher but it is only marginally sensitive to induction by cytokines. Adhesion to both cell types is Ca2+ and Mg2+ dependent. Mitogen-activated lymphocytes bind more strongly to both endothelia, but adhesion to aortic endothelium is not enhanced further by activation of the endothelium. The observed low binding of lymphocytes to brain endothelium and its rapid induction by cytokines suggest a mechanism to explain why lymphocyte accumulation in brain is normally very low but rapidly increases during immune responses. Both cell types express similar levels of class I major histocompatibility complex (MHC) molecules, and this is enhanced by IFN gamma with similar responsiveness to different levels of IFN gamma. MHC class II molecules are absent from these cells but may be induced: although both endothelia respond to similar levels of cytokines, the surface density induced on brain endothelium is approximately 2- to 3-fold higher at all levels of IFN gamma.
We have examined the responses of brain endothelium from different rat strains to interferon-gamma (IFN-gamma) which induces expression of MHC class II molecules. There is a marked difference between the strains in the sensitivity of their endothelium to MHC induction. LEW and DA rats, which are susceptible to experimental allergic encephalomyelitis (EAE) can be induced to express higher levels of class II molecules than EAE-resistant strains. In both the EAE-susceptible and -resistant strains, RT-1B locus-encoded molecules occur at higher surface densities than RT-1D locus molecules. These findings support the theory that genes controlling the induction of MHC expression affect disease susceptibility.