Thymic atrophy in murine acute graft-versus-host disease is effected by impaired cell cycle progression of host pro-T and pre-T cells.

Reconstitution of the peripheral T-cell compartment is a critical aspect for the success of bone marrow transplantation and is also dependent on the reestablishment of normal thymic structure and function. Graft-versus-host disease (GVHD), however, exacerbates posttransplant immunodeficiency through a deleterious effect on thymic function. To investigate the mechanisms of GVHD-mediated thymic disease, 2 murine parent-->F(1 )transplantation models of acute and chronic GVHD, respectively, were studied. Acute GVHD was associated with changes in thymic architecture and a reduction in cellularity mainly because of the decrease in CD4(+)CD8(+), or double-positive (DP) thymocytes, to less than 15% of values found in mice without GVHD. Simultaneously, mature donor-derived T cells expanded in the confines of the allogeneic thymic microenvironment, leading to local inflammation. Through analysis of in vivo cell proliferation, we demonstrated that the ensuing depletion of DP thymocytes was secondary to a decreased commitment of resident pro-T and pre-T cells to enter the cell cycle. Moreover, DP cells themselves showed altered proliferative capacities in the presence of acute GVHD. These findings suggested that thymic atrophy in acute GVHD is effected by impaired cellular proliferation of immature host thymocytes and that the failure of these cells to enter the cell cycle is dependent on an interferon (IFN)-gamma-driven immune response. In contrast, interleukin-4-driven chronic GVHD was not accompanied by a sustained thymic infiltration of donor T cells. Consequently, there was a lack of apparent structural changes, a restricted in situ transcription of inflammatory cytokines, and a virtually unchanged cell cycle progression in vivo.

CD4(+) T cell subsets during virus infection. Protective capacity depends on effector cytokine secretion and on migratory capability.

To analyze the antiviral protective capacities of CD4(+) T helper (Th) cell subsets, we used transgenic T cells expressing an I-A(b)-restricted T cell receptor specific for an epitope of vesicular stomatitis virus glycoprotein (VSV-G). After polarization into Th1 or Th2 effectors and adoptive transfer into T cell-deficient recipients, protective capacities were assessed after infection with different types of viruses expressing the VSV-G. Both Th1 and Th2 CD4(+) T cells could transfer protection against systemic VSV infection, by stimulating the production of neutralizing immunoglobulin G antibodies. However, only Th1 CD4(+) T cells were able to mediate protection against infection with recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G). Similarly, only Th1 CD4(+) T cells were able to rapidly eradicate Vacc-IND-G from peripheral organs, to mediate delayed-type hypersensitivity responses against VSV-G and to protect against lethal intranasal infection with VSV. Protective capacity correlated with the ability of Th1 CD4(+) T cells to rapidly migrate to peripheral inflammatory sites in vivo and to respond to inflammatory chemokines that were induced after virus infection of peripheral tissues. Therefore, the antiviral protective capacity of a given CD4(+) T cell is governed by the effector cytokines it produces and by its migratory capability.

Chemokines and chemotaxis of leukocytes in infectious meningitis.

Chemokines constitute a constantly growing family of small inflammatory cytokines. They have been implied in many different diseases of the CNS including trauma, stroke and inflammation, e.g., multiple sclerosis. In this review we focus on the role of chemokines in infectious meningitis of bacterial or viral origin. In experimental bacterial meningitis induced by Listeria monocytogeneses both CXC and CC chemokines namely MIP-1alpha, MIP-1beta and MIP-2 are produced intrathecally by meningeal macrophages and leukocytes which infiltrate into the CNS. In patients with bacterial meningitis, IL-8, GROalpha, MCP-1, MIP-1alpha and MIP-1beta are detectable in the CSF. These chemokines contribute to CSF mediated chemotaxis on neutrophils and PBMC in vitro. In viral meningitis IL-8, IP-10 and MCP-1 are identified in the CSF to be responsible for chemotactic activity on neutrophils, PBMC and activated T cells. Taken collectively these data indicate that the recruitment of leukocytes in infectious meningitis involves the intrathecal production of chemokines.

The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig.

Integrin-mediated adhesion to the vascular endothelium is an essential step in leukocyte diapedesis. We show that the chemokines 10-kDa inflammatory protein (IP10) and monokine induced by IFN (Mig) induce rapid and transient adhesion of human IL-2-stimulated T lymphocytes (IL-2 T cells) to immobilized integrin ligands through their receptor CXCR3, which is selectively expressed on activated T cells. Induction of adhesion by IP10 and Mig was already observed at subnanomolar concentrations and was maximal at 5-10 nM, resulting in three- to sixfold increase in adhesion of IL-2 T cells over background. No effect was seen with resting naive/memory T cells which lack CXCR3 and migration responses to IP10 and Mig. Both chemokines are produced in human umbilical vein endothelial cells (HUVEC) upon stimulation with IFN-gamma and TNF-alpha. These chemokines induce IL-2 T cell adhesion also when captured on the surface of endothelial cells. Under conditions of flow, IL-2 T cells roll and rapidly adhere to IP10/Mig-expressing HUVEC, and anti-CXCR3 mAb treatment reduces arrest and firm adhesion. This is the first study that shows chemokine-induced adhesion in activated memory/effector T cells which represent the fraction of T cells that are selectively mobilized in inflammation. The critical role of IFN-gamma as inducer of IP10/Mig production in HUVEC indicates that these chemokines are essential mediators of effector T cell recruitment to IFN-gamma-dependent pathologies.

Lymphocyte responses to chemokines.

Today, almost three dozen human chemokines have been identified. The main function of these soluble proteins is the recruitment of leukocytes to sites of infection and inflammation. This review emphasizes the new developments in the field of lymphocyte responses to chemokines. Notably, it was shown that lymphocytes require stimulation to become responsive to chemokines, a process that is closely linked to chemokine receptor expression. As an exception, one chemokine, SDF-1, is a highly effective chemoattractant for non-activated T lymphocytes and progenitor B cells. Of particular interest are the chemokines IP10 and Mig which bind to a receptor with selective expression in activated T lymphocytes and, therefore, may be critical mediators of T lymphocyte migration in T cell-dependent immune-responses. All other chemokines with activities in lymphocytes do also induce responses in monocytes and granulocytes. The involvement of chemokine receptors in HIV infection is briefly mentioned, while other interesting areas in chemokine research, such as hematopoiesis and angiogenesis, are not discussed.

Chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon-gamma inducible protein-10 and monocyte chemotactic protein-1.

In viral meningitis the inflammatory response involves activated T cells and monocytes which are recruited into the subarachnoid space. To identify the chemotactic signals attracting the cells to the site of infection in the meninges, we measured the levels of two CXC chemokines, interferon-gamma (IFN-gamma) inducible protein (IP)-10 and monokine induced by IFN-gamma, four CC chemokines, monocyte chemotactic protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, as well as the cytokines interleukin (IL)-15 and IL-16 in the cerebrospinal fluid (CSF) of patients suffering from viral meningitis. The results point to an involvement of two chemokines, MCP-1 and IP-10, since (1) unlike the other cytokines, MCP-1 and IP-10 were present in 97% and 79% of the CSF, respectively, at concentrations sufficient to induce chemotaxis of mononuclear cells; (2) more than 90% of the CSF of viral meningitis induced chemotaxis of peripheral blood mononuclear cells (PBMC) and all of them induced chemotaxis of activated T cells, and (3) the CSF-mediated chemotaxis of PBMC was inhibited by anti-MCP-1 antibodies and chemotaxis of activated T cells was abolished by the combination of anti-MCP-1 and anti-IP-10 antibodies. Our data provide evidence that MCP-1 and IP-10 lead to accumulation of activated T cells and monocytes in the CSF compartment in viral meningitis.

Cross talk between alpha(v)beta3 and alpha4beta1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1.

Local inflammation leads to increased expression of the vascular cell adhesion molecule (VCAM)-1 on vascular endothelium which contributes to the encapture of leukocytes from the circulating blood through the leukocyte ligand alpha4beta1 integrin. Inflammatory vascular endothelium expresses VCAM-1 at high density. We found that the speed of locomotion of activated lymphocytes migrating along surfaces coated with recombinant VCAM-1 at a comparable density to that found on inflammatory endothelium was slow. However, lymphocytes do migrate and extravasate rapidly under inflammatory conditions, indicating that there must be mechanisms that regulate the interaction between alpha4beta1 and VCAM-1 in vivo. Here we show that the lymphocyte alpha(v)beta3 integrin and integrin-associated protein (IAP) is able to regulate this interaction. The occupancy of lymphocyte alpha(v)beta3 integrin by platelet cell adhesion molecule-1 or vitronectin regulated the speed of alpha4beta1 integrin-dependent locomotion of lymphocytes on recombinant VCAM-1. This allowed rapid lymphocyte migration at VCAM-1 densities which are typical of inflammatory vessels. This alpha(v)beta3-mediated enhanced migration of lymphocytes via alpha4beta1 is likely to depend on the interaction of alpha(v)beta3 integrin with the IAP. Furthermore, this motile process correlates with polarization of the actin cytoskeleton in lymphocytes. Our results suggest that cross talk between alpha(v)beta3 integrin and alpha4beta1 integrin is a mechanism in the regulation of lymphocyte locomotion along inflammatory endothelium and subsequent transendothelial migration. This can explain how lymphocytes overcome tight adhesion to the vascular endothelium and start rapid migration along and through the endothelial lining of blood vessels into inflammatory tissue.

To stick or not to stick: the new leukocyte homing paradigm.

The immune system is formed by leukocytes. They are passively transported through the body by the vascular system, but their entrance into tissues requires a coordinated series of events, namely activation of leukocyte integrins, adhesion to the vascular endothelium, and migration. There are four steps in this process, which begin with the rolling of leukocytes along the vascular endothelium, followed by signaling which activates leukocyte integrins, thus leading to tight adhesion to the endothelium and finally transmigration. Substantial progress has been made recently in elucidating the molecular events that induce rolling and signaling, partly as a result of the study of double-knockout mice that are deficient for genes encoding two selectins.

Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes.

A human receptor that is selective for the CXC chemokines IP10 and Mig was cloned and characterized. The receptor cDNA has an open reading frame of 1104-bp encoding a protein of 368 amino acids with a molecular mass of 40,659 dalton. The sequence includes seven putative transmembrane segments characteristic of G-protein coupled receptors. It shares 40.9 and 40.3% identical amino acids with the two IL-8 receptors, and 34.2-36.9% identity with the five known CC chemokine receptors. The IP10/Mig receptor is highly expressed in IL-2-activated T lymphocytes, but is not detectable in resting T lymphocytes. B lymphocytes, monocytes and granulocytes. It mediates Ca2+ mobilization and chemotaxis in response to IP10 and Mig, but does not recognize the CXC-chemokines IL-8, GRO alpha, NAP-2, GCP-2. ENA78, PF4, the CC-chemokines MCP-1, MCP-2, MCP-3, MCP-4, MIP-1 alpha, MIP-1 beta. RANTES, 1309, eotaxin, nor lymphotactin. The exclusive expression in activated T-lymphocytes is of high interest since the receptors for chemokines which have been shown so far to attract lymphocytes, e.g., MCP-1, MCP-2, MCP-3, MIP-1 alpha, MIP-1 beta, and RANTES, are also found in monocytes and granulocytes. The present observations suggest that the IP10/Mig receptor is involved in the selective recruitment of effector T cells.

CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium.

To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM-1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.

Endothelial vascular cell adhesion molecule 1 expression is suppressed by melanoma and carcinoma.

Vascular cell adhesion molecule 1 (VCAM-1) mediates extravasation of circulating leukocytes into inflamed tissues, and presumably, plays a role in the immigration of cytotoxic effector lymphocytes into tumor metastases. Since metastases are rarely cleared by blood-borne cells from the immune system, we asked whether the tumor may escape host defense by interfering with the mechanism of effector cell extravasation. Here we show that in mice and humans, VCAM-1 expression is repressed on tumor-infiltrating vascular endothelial cells in the lungs. On lung blood vessels distant from the tumor, VCAM-1 is constitutively expressed. When melanoma and endothelioma cells were cultured on either side of a Nucleopore membrane, the expression of VCAM-1 on the endothelioma cells was inhibited and VCAM-1 gene transcription was suppressed. We propose that the downregulation of VCAM-1 is a mechanism by which vascularized melanoma and carcinoma avoid invasion by cytotoxic cells of the immune system.

Murine platelet endothelial cell adhesion molecule (PECAM-1)/CD31 modulates beta 2 integrins on lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells are able to colonize sites of tumor lesions in mouse and man. The molecular mechanisms of homing in on tumors are largely unknown. However, before LAK cells can reach the tumor, they must adhere to the vascular endothelial within the lesion and then extravasate. We developed a novel mAb, EA-3, which recognizes the murine homologue of the human adhesion molecule CD31. It is present on a subpopulation of murine LAK cells and all endothelial cells. CD31 was also involved in the adhesion of LAK cells to endothelium. Since CD31 can initiate integrin activation by inside-out signaling after binding to its ligand, EA-3 was used to minimic this in adhesion assays. It induces modifications in the beta 2 integrin LFA-1, leading to increased binding capacities of the cells to endothelium. In contrast, beta 1 integrins and RGD-binding integrins were not affected. These results suggest that expression of CD31 might confer adhesive advantages for LAK cells prone to tumor infiltration.