Resistance of Crohn's disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance.

Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2+, and Bax+ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-alpha and nitric oxide. TUNEL+ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68+ and myeloperoxidase+, but no CD45RO+, apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivation-induced apoptosis. CD T cells were also more resistant to Fas- and nitric oxide-mediated apoptosis, whereas TNF-alpha failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2+, but fewer Bax+, cells, while UC mucosa contained fewer Bcl-2+, but more Bax+, cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.

In vivo cytokine and neuroendocrine responses to endotoxin in human immunodeficiency virus-infected subjects.

The cytokine and neuroendocrine host responses to experimental challenge with lipopolysaccharide (LPS) were studied in human immunodeficiency virus (HIV)-infected subjects and uninfected control subjects. Elevations in circulating concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 were significantly greater in HIV-infected subjects than control subjects after LPS challenge. All subjects showed a significant increase in circulating concentrations of adrenocorticotropin, cortisol, and norepinephrine after LPS challenge, but there was not a significant difference between the responses of these hormones in the HIV-infected and -uninfected subjects. Compared with the control subjects, the HIV-infected subjects had a significantly reduced IL-10 response and a reduced IL-1 receptor antagonist response. It is concluded that the TNF-alpha, IL-6, IL-8, and IL-10 cytokine responses to LPS in vivo are disrupted in HIV subjects but that this is not related to disruption of the hypothalamo-pituitary-adrenal axis.

Integrative aspects of a human model of endotoxemia.

The production of tumor necrosis factor (TNF)-alpha is a key step in the response to sepsis and has powerful local and systemic effects on the host. These systemic responses include a complex cascade of centrally mediated endocrine and neural responses. An integrative model of these regulatory cytokine-neuroendocrine interactions in humans is presented. The rapid kinetics of these responses are illustrated by data showing the response of normal human subjects to experimental endotoxemia. Appreciation of the integrative biology of the in vivo response to experimental endotoxemia can provide a framework for the design of experiments aimed at examining the effects of physical training paradigms on particular cytokine and neuroendocrine pathways.

The effect of exercise on lymphocyte redistribution and leucocyte function in asymptomatic HIV-infected subjects.

This study was undertaken to examine the responsiveness of circulating leucocyte and lymphocyte populations to the physiological demands of exercise in asymptomatic HIV-infected subjects with CD4+ counts greater than 500/microliter. Thirteen subjects infected with HIV and 14 control subjects underwent 20 min of defined moderate exercise at estimated 65% of their maximal ventilatory capacity on a bicycle ergometer. Blood samples were obtained for serum cortisol, norepinephrine, lymphocyte subsets (CD4, CD8, CD19, CD16-CD56), and phagocytic function at rest immediately after exercise and 20 min following the cessation of the exercise. The HIV-infected subjects had increased circulating concentrations of CD8 cells (p = .007) and CD16-CD56+ NK cells (p = .02) in response to the exercise, whereas the control group did not. There was a greater increase in monocyte respiratory burst activity following recovery from exercise in the control subjects (p = .016) but not in the HIV-infected subjects. The control subjects experienced an increase in serum cortisol in response to the exercise (p = .006), but the HIV-infected subjects did not. Our results show that the changes in the distribution and function of circulating leucocytes and adrenal neuroendocrine responses to moderate exercise differ in asymptomatic HIV-infected and control subjects.

Frequent medical visits by cocaine-using subjects in a Canadian community: an invisible problem for health practitioners?

The purpose of this study was to examine the frequency of medical visits by cocaine-using subjects in a Canadian community. A sample of 100 subjects reporting cocaine use at least 10 times in the previous 12 months were recruited in an urban setting in Canada and interviewed in a structured manner to address aspects of their use of cocaine and their responses to those agents. The respondents reported a total of 488 medical visits in the 12 months prior to interview. The frequency of visits correlated with the use of cocaine, barbiturates, hallucinogens, narcotics, and amphetamines. Medical visits also varied with the frequency with which the subjects reported certain adverse reactions to cocaine. Logistic regression modeling was used to assign subjects into a higher medical contact group (three or more medical visits per 12 months) and a lower medical contact group (two or fewer medical visits per 12 months). Membership in the higher or lower contact group was differentiated by a simple model in which the classifying variables were whether or not the subjects reported using crack cocaine in the previous year, whether or not they reported using hallucinogens in the previous year, and whether or not they reported experiencing aggressive reactions with the use of cocaine. Thus, users of cocaine report frequent visits to physicians. Medical visits are more likely if they also used crack cocaine, if they experienced aggressive reactions to cocaine, and if other substances were also used. Recognition of this behavior may facilitate earlier intervention by primary care physicians.

Role of the neuroendocrine system in cytokine pathways in inflammatory bowel disease.

The intestine is a rich venue for interactions between the neuroendocrine and immune systems. Functional regulation of mucosal immune responses can be exerted through local neuroendocrine paths by neuropeptides from the neurons of the enteric nervous system and peptide hormones. Immune regulation can also be exerted through influences in the central nervous system activating the hypothalamic-pituitary-adrenal axis, and through the activation of the autonomic nervous system. The integrity of these systems can alter the outcome of immune-mediated inflammation in the gut. The role of these pathways in the regulation of cytokines is examined, and the implications for Crohn's disease and ulcerative colitis are explored.

The influence of neuroendocrine pathways on lymphocyte migration.

The immunological work that leads to the production of effector cells, immunoglobulins and cytokines in intact animals results from the coordinated interaction of clusters of specialized lymphocytes. These lymphoid clusters function in microenvironments within which they may be exposed to neural and endocrine signals, and the ability of such signals to modulate the local output of immune labor is now well recognized. Here, Clifford Ottaway and Alan Husband review evidence suggesting that the output of neuroendocrine pathways has a modulatory effect on the migratory behavior of lymphocytes in vivo. This can lead to rapid changes in the specific phenotypes of lymphocytes accumulating in tissues and organs undergoing immune challenge.

Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection.

The induction of monocyte/macrophage procoagulant activity (PCA) has been implicated in the pathogenesis of murine hepatitis virus strain 3 (MHV-3) infection and disease. Previously, we have shown that induction of PCA by MHV-3 correlated with resistance/susceptibility to infection in different mouse strains. In this study, all BALB/cJ mice that were infected with 10(3) plaque-forming units of MHV-3 developed severe liver disease and died within 96-120 h. Examination of the livers of these animals showed marked hepatic necrosis, deposition of fibrin, and cellular expression of PCA by direct immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. Splenic mononuclear cells recovered from these mice expressed high concentrations of PCA with time after infection. Infusion into mice of a high-titered monoclonal antibody that neutralized PCA (3D4.3) attenuated the development of hepatic necrosis and enhanced survival in a dose-dependent manner. All of the animals receiving 100 micrograms, and 44% and 22% of the animals that received 50 and 25 micrograms per day, respectively, survived for 10 d and made a full recovery. Administration of the antibody resulted in a dose-dependent reduction in fibrin deposition, PCA expression as detected by direct immunofluorescence staining and by a functional assay. In animals treated with high concentrations of antibody, titers of antibody to PCA fell from 87 +/- 15 micrograms/ml to 100 +/- 7 ng/ml during the active phase of the disease, consistent with sequestration due to binding of the immunoglobulin to cells expressing PCA. Surviving animals, when rechallenged with MHV-3, had a 40% mortality, consistent with the known rates of metabolism of immunoglobulin. This further suggested that protection was by a passive mechanism. The results reported here demonstrate that a neutralizing antibody to PCA protects animals from fulminant hepatitis and death associated with MHV-3 infection, and supports the notion that PCA is a potent inflammatory mediator that plays a pivotal role in the pathogenesis of liver injury resulting from MHV-3 infection.

Insertion and internalization of vasoactive intestinal peptide (VIP) receptors in murine CD4 T lymphocytes.

The specific binding of vasoactive intestinal peptide (VIP) to murine lymphocytes was investigated. CD4 T cells from mesenteric lymph nodes (MLN) bound more 125I-VIP than did unseparated MLN lymphocytes at 37 degrees C, but not at 4 degrees C. The differences between the amount of 125I-VIP bound by the CD4 T cells and unseparated MLN lymphocytes at 37 degrees C depended upon a difference in the amount of the ligand that was internalized by the cells. The rate of insertion of unoccupied VIP receptors from the cytoplasm into the cell membrane (370 receptors/cell/min), the rate constants for internalization of ligand occupied VIP receptors (0.55 min-1) and unoccupied VIP receptors (0.11 min-1), and the rate constant for the elimination of internalized VIP (0.07 min-1) by CD4 T cells were evaluated. These results provide new understanding of the behaviour of VIP receptors on lymphocytes and indicate a mechanism by which CD4 T lymphocytes can homologously regulate their surface expression of VIP receptors in the presence of ambient VIP.

Role of sulfhydryl groups in the binding of vasoactive intestinal peptide to its receptor on murine lymphocytes.

The effect of sulfhydryl-containing compounds on the specific binding of the neuropeptide vasoactive intestinal peptide (VIP) to murine lymphocytes was studied. Both 2-mercaptoethanol (2ME) and dithiothreitol (DTT) inhibited VIP-binding to lymphocytes at millimolar concentrations. A sulfhydryl-containing analogue of VIP, [Cys2]VIP, was synthesized. This compound competed for the binding of [125I]VIP about 150,000 x more effectively than 2ME, but was approximately 100 x less effective than VIP itself. Both VIP and [Cys2]VIP increased intracellular cyclic AMP and inhibited the proliferative response of lymphocyte cultures to concanavalin A (ConA), but the molar potency of [Cys2]VIP on these lymphocyte activities was approximately 100 x less than that of VIP. The effects of VIP and [Cys2]VIP on intracellular cyclic AMP and ConA-stimulated proliferation were competed for by the VIP receptor antagonist [4Cl-D-Phe6,Leu17]VIP. Replacement of serine2 with L-cysteine disrupts the ability of VIP to occupy and activate lymphocyte VIP receptors. This may reflect a role of serine2 in hydrogen-bond formation during ligand-receptor interactions, or a functional role of sulfhydryl-containing residues of the VIP receptor in maintaining the integrity of the binding site of the VIP receptor on lymphocytes.

Central nervous system influences on lymphocyte migration.

The immune response network is only one of many physiologic adaptive responses to environmental change and there is now substantial evidence that adaptive responses involving the central nervous system have an impact on immune outcome. Effective immune function depends upon a highly mobile population of precursor and effector cells of the lymphoid system. In this review it is proposed that many of the alterations in immunity resulting from CNS activity may be explained in terms of changes in lymphocyte migration patterns in response to endocrine signals, neural signals via neurotransmitter release, or direct contacts between nerves and cells of the immune system.

Receptors for vasoactive intestinal peptide on murine lymphocytes turn over rapidly.

The interaction of the neuropeptide vasoactive intestinal peptide (VIP) with its receptors on murine mesenteric lymph node lymphocytes (MLN) has been re-examined in detail. Intact MLN actively internalize surface bound VIP. The rate constants associated with the insertion of receptors at the MLN surface, the internalization of VIP occupied and unoccupied receptors and the elimination of the peptide were determined. At 37 degrees C, MLN insert approximately 140 VIP receptors cell-1 min-1 at their surface, and the rate of internalization of occupied receptors (0.23 min-1) was much greater than that of the unoccupied receptors (0.02 min-1). Exposure of MLN to non-saturating concentrations of VIP markedly altered the expression of VIP receptors at the lymphocyte surface. The rapid turnover of VIP receptors combined with the differential clearance of occupied and unoccupied receptors from the cell surface provides a mechanism by which homologous regulation of VIP receptor expression can occur on these lymphocytes.

Colonic vasoactive intestinal peptide nerves in inflammatory bowel disease.

Vasoactive intestinal peptide is a neuropeptide with potent modulatory activity on intestinal immunity and may be implicated in the pathogenesis of inflammatory bowel disease (IBD). Previous studies have reported abnormal morphology of vasoactive intestinal peptide-stained enteric nerves, in addition to increased, normal or decreased levels of extractable peptide in Crohn's disease (CD) and ulcerative colitis (UC) tissues. These observations have not been correlated with the amount of enteric nerve fibers or the degree of mucosal inflammation. The investigation was intended to determine whether abnormalities of vasoactive intestinal peptide in IBD are related to quantitative changes of enteric nerve fibers or mucosal inflammation, and whether they are specific for CD or UC. To do this, digitized morphometric analysis was applied to a large number of IBD and control colonic surgical specimens that were immunostained for vasoactive intestinal peptide and S100 protein and scored for severity of inflammation. The results showed that, as compared with controls, there is a marked decrease of vasoactive intestinal peptide-immunoreactive nerve fibers in the lamina propria and submucosa (P less than 0.0001), and of S100-immunoreactive nerve fibers in the lamina propria (P less than 0.0001) of patients with IBD. In the lamina propria but not the submucosa, the variation of decrease is significantly associated with the severity (P less than 0.0001) but not the type (P greater than 0.9) of IBD because it is detected in both CD and UC. We conclude that in IBD there is loss of mucosal neuropeptidic innervation that is intimately associated with inflammation. This loss probably represents a nonspecific event subsequent to damage to enteric nerve fibers but may contribute to disruption of local immunoregulation.

Gene replacement with one-sided homologous recombination.

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

Neuroimmunomodulation in the intestinal mucosa.

The intestine contains major subdivisions of the nervous and immune systems. The lymphoid compartments of the intestine contain functionally distinguishable populations of immunologic cells and are innervated differently. The lamina propria has an extensive network of nerves using the neuropeptides SOM, SP, and VIP. Subpopulations of T cells, B cells, cells of the monocyte/macrophage line, and several other immunologically relevant cells have the ability to recognize and respond to these neuropeptide signals. SOM, SP, and VIP can act as potent regulators of lymphoid cell proliferation and interleukin and immunoglobulin production. The unusual effector lymphocytes in the epithelial layer of the intestine can be exposed to SP and VIP, and their responses may be regulated by these peptides. In the organized lymphoid compartments such as Peyer's patches, the neuropeptides VIP and SP may regulate the accumulation or recirculation of affector lymphocytes from the central compartment of the immune system and their subsequent response to antigens. The large array of immunoregulatory effects that have been found with these neuropeptides suggest that local neurophysiologic signals in the intestinal lymphoid microenvironments can regulate selected aspects of immune responses. The intestine is likely to be a highly specialized venue for neuroimmunomodulation in intact animals, and this has important implications in the physiologic and pathologic responses of the gut. Further investigations of these regulatory pathways will lead to new concepts concerning neural-immune interactions in general and the regulation of mucosal immunology in particular.

High affinity specific binding of vasoactive intestinal peptide to human circulating T cells, B cells and large granular lymphocytes.

We studied the specific binding of vasoactive intestinal peptide (VIP) to circulating lymphocytes (PBL) of normal subjects using the interaction of 125I-VIP with different PBL fractions and flow cytometry to detect the binding of VIP-coated polystyrene spheres to individual cells of the fractions. Enhanced binding of 125I-VIP was found with T-enriched compared to unseparated or T-depleted PBL preparations. Both CD4 and CD8 T cell-enriched suspensions showed high binding capacity, but the affinity of CD4-enriched preparation for VIP was higher. VIP-coated spheres also bound to individual T cells of PBL, but only a minority of CD4 T cells (32%) and CD8 T cells (23%) bound the spheres. 125I-VIP also specifically bound to fractions enriched for large granular lymphocytes (LGL) and B cells. A consistent proportion of CD16 marker-positive LGL bound VIP-coated spheres (24%, and approximately 15% of B cells also showed this ability. Thus there is marked heterogeneity in the ability of different phenotypes of normal human circulating lymphocytes to recognize this neuropeptide.

The kinetics of migration of murine CD4 and CD8 lymphocytes in vivo.

Lymphocytes from the mesenteric lymph nodes (MLN) of mice were enriched for CD4+ and CD8+ T cell populations, labeled with (51Cr) sodium chromate, and transferred to the bloodstream of syngeneic recipients. The time course of migration of the labeled cells from the blood to the secondary lymphoid organs of the recipients was investigated. CD4+ and CD8+ subpopulations differed in their profile of appearance in different lymphoid organs. Computer assisted analysis of the observations was used to obtain quantitative estimates of the rate of clearance of the lymphocytes from the blood into the tissues, and the rate of departure of the cells from the tissues. In the spleen, CD4+ lymphocytes were cleared from the blood about one and one-half times faster than CD8+ lymphocytes, but the CD8+ cells were retained longer. Inguinal nodes (IN), MLN, and Peyer's patches (PP) showed a consistent ability to clear CD4+ cells from the blood at a rate approximately 2.5 x greater than that for CD8+ cells, but the retention of the lymphocytes in these tissues varied with lymphocyte phenotype and the organ concerned. CD4+ lymphocytes were retained longer in PP and MLN than in IN, whereas CD8+ cells were retained longer in IN and MLN nodes than in PP. We conclude that the rate of clearance of lymphocytes into secondary lymphoid organs from the blood varies in a regular way with T cell phenotype and that organ specific sorting of T subpopulations also proceeds after the cells are admitted to the tissues.