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The effect of peripheral immunization with Mls-1a on the emigration of antigen-specific cells from the thymus.

Mature T cells found in the lymph nodes and spleen have the capacity to become activated and to proliferate in response to foreign antigens. The response of the thymus to such immunization is less well understood. We have examined one aspect of the thymic response by determining the effect of peripheral immunization upon cell emigration from the thymus. BALB/c (Mls-1b) mice were injected with spleen cells from DBA/2 (Mls-1a) mice, and V beta 6+ (Mls-1a-reactive) thymic emigrants were identified 3-30 days after immunization. Neither the rate of total cell migration from the thymus nor the proportion of V beta 6+ cells was altered, even though the immunizing spleen cells elicited an immune response in the draining (parathymic) lymph nodes. The same immunogen caused deletion of V beta 6+ cells in both the thymus and lymph nodes after intraperitoneal injection into the neonate. The inability of DBA/2 splenocytes to modify the development of adult thymocytes after intrathymic injection of the cells precluded the lack of entry into the thymus as the reason for the lack of any observed effect in the adult. Our results, therefore, indicate that the development of adult thymocytes is not modified by immunization, and suggest that the differing thymic response of mice injected as adults or neonates is related to changes in the intrathymic antigen presentation capacity associated with age.

Age Factors↗

Role of the aryl hydrocarbon receptor in thymocyte emigration in vivo.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent member of the PAS-bHLH-family of nuclear receptors. Anthropogenic ligands include environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Over-activation of the AHR causes thymus atrophy and immunosuppression. Signaling via the AHR changes the thymocyte differentiation program at several checkpoints, in particular within the CD4-CD8- double-negative (DN) thymocyte subset. Here, we show that AHR over-activation led to the preferential emigration of DN thymocytes to the periphery and accumulation in the spleen. Some of these recent thymic emigrants (RTE) had a novel "activated immature" phenotype (CD3-TCRbeta-CD25+/intCD44-CD45RB+/intCD62L+CD69- cells). Gene expression profiling of DN RTE revealed 15 genes that were up-regulated more than threefold by TCDD, including the S100A9 gene. Exposure of S100A9 null mice to TCDD showed a role for this protein in AHR-mediated thymic egress.

Animals↗

The role of PECAM-1 (CD31) in leukocyte emigration: studies in vitro and in vivo.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a molecule capable of mediating both homophilic and heterophilic adhesion. It is constitutively expressed and concentrated in the lateral borders between endothelial cells and expressed on the surfaces of neutrophils, monocytes, and some T cell subsets, as well as on platelets. In a quantitative in vitro assay, monoclonal antibody against PECAM-1 or soluble recombinant PECAM-1 selectively blocked passage of both neutrophils and monocytes across the endothelial monolayer by 70-90% without interfering with the ability of these cells to bind to the apical endothelial cell surface. These regents worked whether directed against leukocyte PECAM-1 or against endothelial cell PECAM-1 and were not additive, suggesting that a homophilic interaction was occurring. In a murine model of acute inflammation, thioglycollate-induced peritonitis, a monoclonal antibody against mouse PECAM-1 blocked emigration of leukocytes into the peritoneal cavity down to background levels. Examination of peritoneal venules in these mice revealed many leukocytes in apparent contact with the endothelial surface but unable to cross the intima. Thus, PECAM-1 has a distinct role in the transendothelial migration phase of leukocyte emigration, independent of the adhesion events on the apical surface.

Animals↗

Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants.

Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.

Alleles↗

A low molecular weight component derived from the milk of hyperimmunised cows suppresses inflammation by inhibiting neutrophil emigration.

An earlier study demonstrated that hyperimmunisation of dairy cows with a polyvalent bacterial vaccine stimulated the secretion of a small molecular weight anti-inflammatory moiety in the milk. This hyperimmune milk factor (HIMF) has been further investigated in the present experiments. HIMF was found to suppress the cellular phase of the response to carrageenin and also the neutrophil-dependent reverse passive Arthus reaction. These results, together with the observation that the administration of HIMF led to an increase in the number of circulating neutrophils, suggested that the agent might inhibit inflammation by interfering with the ability of neutrophils to emigrate from the vasculature. In vivo studies carried out to evaluate this possibility demonstrated that HIMF suppressed neutrophil emigration by up to 75%. In vitro experiments established that the ability of neutrophils to respond to chemotactic stimuli or adhere to endothelial cells was not affected by HIMF. It is possible, therefore, that the agent modulates inflammation by down-regulating the synthesis of inducible pro-inflammatory cytokines or adhesion molecules. Attempts are presently being made to isolate the active moiety to allow the activity of the agent at the molecular level to be studied in more detail.

Animals↗

Effects of SC-41930 on leukocyte adherence and emigration in rat mesenteric venules.

This study demonstrates that SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8-p ropyl-2H-1-benzopyran-2-carboxylic acid, an orally active LTB4 receptor antagonist, reduces LTB4-induced leukocyte adhesion and emigration in rat mesenteric venules. The mesentery of Sprague-Dawley rats was prepared for intravital microscopic examination and venules of 25-35 microns were chosen for evaluation. In control animals, LTB4 (20 nM) was superfused over the mesentery for 30 min. In the treatment group SC-41930 (5 microM) was superfused for 30 min, followed by a 30 min superfusion with SC-41930 and LTB4. The LTB4-induced increase in leukocyte adherence and emigration in postcapillary venules was significantly attenuated by pretreatment with SC-41930. Other experiments demonstrated that platelet-activating-factor-induced leukocyte adherence was not affected by SC-41930. These results indicate that SC-41930 is a potent inhibitor of LTB4-induced leukocyte-endothelial cell adhesive interactions in postcapillary venules.

Animals↗

Dextran sulphate inhibits neutrophil emigration and neutrophil-dependent plasma leakage in rabbit skin.

Rolling of neutrophils in postcapillary venules is believed to be an obligatory step in the adhesion of neutrophils to endothelial cells, preceding firm attachment and emigration. This rolling on the endothelial surface can be blocked by sulphated polysaccharides. Therefore, dextran sulphate or dextran T500 was injected intravenously (i.v., 25 mg/kg) and neutrophil infiltration and plasma leakage in skin were measured in response to intradermal (i.d.) injection of inflammatory mediators in the presence of calcitonin gene-related peptide (CGRP). Neutrophil accumulation and oedema formation induced by the neutrophil chemoattractants, f-Met-Leu-Phe (FMLP) or C5adesArg, were completely suppressed in the presence of dextran sulphate. Neutrophil emigration in response to interleukin-1 (IL-1) was not affected by dextran sulphate. These results suggest that neutrophil recruitment induced by FMLP and C5adesArg, but not by IL-1, is mediated via sulphated glycans, perhaps on endothelial cells.

Animals↗

Selective emigration of suppressor T cells from Peyer's patches.

The emigration of Peyer's patch lymphocytes to mesenteric lymph nodes was studied by injecting fluorescein isothiocyanate (FITC) directly into Peyer's patches. Using double immunofluorescence it was demonstrated that at 2 and 4 hr after FITC injection 70% of the labeled cells that migrated to mesenteric lymph nodes were T lymphocytes, although rat Peyer's patches contain only 15-20% T lymphocytes. At later time points after FITC injection this percentage of T cells derived from Peyer's patches gradually declined, most likely caused by selective interaction and/or retention inside the mesenteric lymph node. Determination of helper and suppressor T-cell subsets within this emigrating population showed an increased number of T suppressor cells migrating into mesenteric lymph nodes. The putative role of suppressor T cells in inducing systemic tolerance after oral antigen administration was discussed.

Animals↗

Private alleles in a partially isolated population. II. Distribution of persistence time and probability of emigration.

Two diffusion limits were derived from a discrete Wright-Fisher model of migration, mutation, and selection with an arbitrary degree of dominance. Instantaneous killing of the process due to emigration of a mutant leads to one of two diffusion processes with a killing term. One (weak gene flow) is the boundary case of the other (strong gene flow), which can cover a wide range of gene flow. The diffusion process subject to strong gene flow is similar to that studied by S. Karlin and S. Tavaré (1983, SIAM J. Appl. Math. 43, 31-41). The spectral decomposition of the transition probability density of "private" allele frequencies is presented in the case of strong gene flow. The fate of mutant in a deme is discussed in terms of the probabilities of survival and emigration.

Alleles↗

Leukotrienes cause eosinophil emigration into conjunctival tissue.

The ability of LTB4, LTC4, the 5S,6R and 5R,6S LTD4 stereoisomers, and LTE4 to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological and light microscopy techniques. LTD4 and LTE4 demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10 ng to 1000 ng), while there was only a minimal response to LTC4. LTB4 produced marked eosinophil infiltrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB4. The 5R,6S LTD4 stereoisomer did not evoke any leukocyte infiltration. The SRS-A antagonist, FPL 55712, abolished peptidoleukotriene-induced eosinophil emigration, and indomethacin pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10 micrograms to 1000 micrograms. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.

Animals↗

Up-regulation of cell surface Toll-like receptor 4-MD2 expression on dendritic epidermal T cells after the emigration from epidermis during cutaneous inflammation.

BACKGROUND: Mouse epidermis contains a population of gammadelta T cells, termed dendritic epidermal T cells (DETCs), which uniformly express the invariant Vgamma3 T cell receptor. Certain DETC lines were reported to respond to Gram-negative bacteria in the presence of immobilized anti-CD3 monoclonal antibody or to lipopolysaccharide (LPS) in the presence of B cell lines. OBJECTIVE: To determine whether DETCs express the primary signaling receptor for LPS, Toll-like receptor (TLR) 4-MD2. METHODS: We analyzed expression of TLR4-MD2 in three independent DETC lines as well as in freshly isolated DETCs. RESULTS: All DETC lines expressed TLR4 and MD2 transcripts and TLR4-MD2 protein complex intracellularly, but none expressed TLR4-MD2 on the cell surface. By immunoblotting, only the immature form of TLR4 protein was detected in the DETC lines. The DETC lines did not respond to LPS even in the presence of immobilized anti-CD3 monoclonal antibody. Freshly isolated DETCs and their fetal thymic precursors also lacked cell surface expression of TLR4-MD2, but a small subpopulation of dermal Vgamma3 T cells isolated from croton oil-painted skin expressed TLR4-MD2 on the cell surface. Similarly, Vgamma3 T cells emigrated from organ-cultured epidermis expressed cell surface TLR4-MD2. CONCLUSIONS: These results demonstrate that DETCs do not constitutively express cell surface TLR4-MD2, but TLR4-MD2 expression may be up-regulated when DETCs emigrate from epidermis during cutaneous inflammation.

Animals↗

Actions of emigrated neutrophils on Na(+) and K(+) currents in rat ventricular myocytes.

Interactions between neutrophils and the ventricular myocardium can contribute to tissue injury, contractile dysfunction and generation of arrhythmias in acute cardiac inflammation. Many of the molecular events responsible for neutrophil adhesion to ventricular myocytes are well defined; in contrast, the resulting electrophysiological effects and changes in excitation-contraction coupling have not been studied in detail. In the present experiments, rat ventricular myocytes were superfused with either circulating or emigrated neutrophils and whole-cell currents and action potential waveforms were recorded using the nystatin-perforated patch method. Almost immediately after adhering to ventricular myocytes, emigrated neutrophils caused a depolarization of the resting membrane potential and a marked prolongation of myocyte action potential. Voltage clamp experiments demonstrated that following neutrophil adhesion, there was (i) a slowing of the inactivation of a TTX-sensitive Na(+) current, and (ii) a decrease in an inwardly rectifying K(+) current. One cytotoxic effect of neutrophils appears to be initiated by enhanced Na(+) entry into the myocytes. Thus, manoeuvres that precluded activation of Na(+) channels, for example holding the membrane potential at -80 mV, significantly increased the time to cell death or prevented contracture entirely. A mathematical model for the action potential of rat ventricular myocytes has been modified and then utilized to integrate these findings. These simulations demonstrate the marked effects of (50-fold) slowing of the inactivation of 2-4% of the available Na(+) channels on action potential duration and the corresponding intracellular Ca(2+) transient. In ongoing studies using this combination of approaches, are providing significant new insights into some of the fundamental processes that modulate myocyte damage in acute inflammation.

Action Potentials↗

Leukocyte emigration in the early stages of laminitis.

The mechanisms that initiate the pathophysiologic changes in the digital laminae in equine laminitis are poorly understood. Due to the fact that (1) the horse at risk of laminitis has many similarities clinically to the human sepsis patient and (2) our recent finding of marked laminar proinflammatory cytokine expression at the developmental time point of the black walnut extract (BWE) model of laminitis, we tested the possibility that, similar to organ damage in human sepsis, leukocyte emigration is an early event in laminitis. Using immunoperoxidase methods with an anti-equine CD13 monoclonal antibody that recognizes neutrophils and monocytes, we discovered that, whereas the dermal microvasculature of the skin commonly has a marginal pool of leukocytes, the normal laminar dermal microvasculature has minimal to no perivascular leukocytes. However, increases in leukocyte numbers occurred around the dermal vasculature of both the laminae and the skin in the majority of BWE-treated horses in the developmental stage and at the onset of clinical signs of lameness in the BWE model. These findings indicate that, similar to organ failure in human sepsis, leukocyte emigration is likely to play a significant role in initiating numerous pathophysiologic mechanisms that lead to the development of laminitis.

Animals↗

Effect of recent thymic emigrants on progression of HIV-1 disease.

BACKGROUND: The concentration of T-cell receptor-rearrangement excision DNA circles (TREC) in peripheral-blood T cells is a marker of recent thymic emigrant alphabeta T cells. We studied the predictive ability of measurements of TREC for clinical outcome in HIV-1-infected individuals. METHODS: We measured TREC in peripheral-blood mononuclear cells with a real-time PCR assay. We studied 131 Greek participants in the Multicenter Hemophilia Cohort Study who had known HIV-1 seroconversion dates. The prognostic value of baseline TREC, CD4 T-cell count, and HIV-1 RNA concentration was assessed by Kaplan-Meier and Cox's regression analysis. FINDINGS: Four participants had progressed to AIDS by first blood sampling. Among the remaining 127 individuals, the median value of TREC per 10(6) cells was 6900 (IQR 2370-15604). Baseline TREC values were lower in the 53 who progressed to AIDS than in those who did not (geometric mean 2843 [95% CI 1468-5504] vs 6560 [4723-9113] per 10(6) cells; p=0.017). The relative hazard of AIDS, adjusted for plasma viral load, CD4 T-cell count, and age at seroconversion was 1.44 (95% CI 1.04-2.01; p=0.031) per ten-fold increase in TREC; that for death was 1.52 (1.12-2.06; p=0.007). The adjusted relative hazards of death were 2.91 (1.91-4.44; p<0.001) per ten-fold increase in plasma HIV-1 RNA load and 1.20 (1.04-1.38; p=0.014) per 100-cell decrease in CD4 T-cell count. INTERPRETATION: The concentration of TREC in the peripheral T-cell pool complements HIV-1 RNA load and CD4 T-cell count in predicting the rate of HIV-1 disease progression. Recent thymic emigrants have a role in the pathogenesis of HIV-1 disease.

Adult↗

The development of humoral immunological memory to a T-cell-dependent antigen requires thymic emigrant cells.

Immunological memory is embodied in the rapid and enhanced immune responsiveness to previously encountered antigens. Classically, memory would depend on the presence of small resting long-lived specific lymphocytes which, through clonal expansion after priming with antigen, would be present at higher frequencies than in naive animals. Here we report that T-cell-reconstituted athymic mice, which lack recent thymic emigrants, mount a primary response to a T-cell-dependent antigen, but do not develop memory or the capacity to produce specific anti-TNP IgG1 antibodies during the secondary immune response. On the other hand, if thymocytes are continuously provided during the secondary response, a typical secondary immune response is achieved with high levels of specific IgG1. These results lead us to propose that the development of humoral immunological memory cannot be explained solely by the long life span of primed T lymphocytes, but is rather a dynamic state dependent on the continuous presence of recent thymic emigrants and qualitative functional differences in responder T cells.

Animals↗

Role for CCR7 ligands in the emigration of newly generated T lymphocytes from the neonatal thymus.

Most T lymphocytes are generated within the thymus. It is unclear, however, how newly generated T cells relocate out of the thymus to the circulation. The present study shows that a CC chemokine CCL19 attracts mature T cells out of the fetal thymus organ culture. Another CC chemokine CCL21, which shares CCR7 with CCL19 but has a unique C-terminal extension containing positively charged amino acids, failed to show involvement in thymic emigration. Neonatal appearance of circulating T cells was defective in CCL19-neutralized mice as well as in CCR7-deficient mice but not in CCL21-neutralized mice. In the thymus, CCL19 is predominantly localized in the medulla including endothelial venules. These results indicate a CCL19- and CCR7-dependent pathway of thymic emigration, which represents a major pathway of neonatal T cell export.

Animals↗

Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2.

Monocytes recruited to tissues mediate defense against microbes or contribute to inflammatory diseases. Regulation of the number of circulating monocytes thus has implications for disease pathogenesis. However, the mechanisms controlling monocyte emigration from the bone marrow niche where they are generated remain undefined. We demonstrate here that the chemokine receptor CCR2 was required for emigration of Ly6C(hi) monocytes from bone marrow. Ccr2(-/-) mice had fewer circulating Ly6C(hi) monocytes and, after infection with Listeria monocytogenes, accumulated activated monocytes in bone marrow. In blood, Ccr2(-/-) monocytes could traffic to sites of infection, demonstrating that CCR2 is not required for migration from the circulation into tissues. Thus, CCR2-mediated signals in bone marrow determine the frequency of Ly6C(hi) monocytes in the circulation.

Animals↗

Blocked B cell differentiation and emigration support the early growth of Myc-induced lymphomas.

Avian leukosis virus induces lymphoma in chickens after proviral integration within the c-Myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed bursal follicles. The clonal expansion of these follicles allowed us to examine how Myc influences cell differentiation, growth, and apoptosis in lymphoid progenitors soon after the onset of Myc overexpression. Immunohistochemical analysis of developmental markers established that Myc overexpression consistently blocks lymphocyte differentiation at a late embryonic stage. Myc-transformed follicles also grow much more rapidly than normal follicles. This rapid growth is not mediated by suppression of apoptosis, as normal and Myc-transformed follicles showed similar rates of cell death by TUNEL immunohistochemical analysis of cells undergoing DNA degradation. Measurements of DNA synthesis and mitotic index showed modest effects of Myc to increase lymphocyte proliferation, as normal lymphocytes already divide rapidly. The major mechanism mediating rapid growth of transformed follicles instead involved failure of myc-overexpressing lymphocytes to emigrate from transformed follicles, while normal lymphocytes actively emigrate after hatching, as measured by BrdU pulse-chase labeling and immunohistochemical measurements. This failure to undergo the normal program of differentiation and subsequent bursal retention of lymphocytes accounts for most of the growth of transformed follicles, while Myc-induced proliferation makes a smaller contribution.

Animals↗