The α4ß1 Homing Pathway Is Essential for Ileal Homing of Crohn's Disease Effector T Cells In Vivo.

BACKGROUND: The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohn's disease (CD) are still unclear, and clinical outcome data from patients with inflammatory bowel disease treated with the anti-α4ß7 integrin antibody vedolizumab suggest differences between ulcerative colitis and CD. METHODS: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo. RESULTS: Despite in vitro blockade of CD Teff adhesion to mucosal vascular addressin cell adhesion molecule-1 (MadCAM-1) and in contrast to previous observations in ulcerative colitis, anti-α4ß7 treatment did not result in reduced Teff cell homing to the colon in vivo. However, the integrin α4ß1 was expressed in higher levels on Teffs from patients with CD compared with controls, while its expression in the peripheral blood declined, and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to vascular cell adhesion molecule-1 (VCAM-1) was blocked by inhibition of α4 and α4ß1 in vitro. Moreover, in vivo homing of CD Teffs to the ileum was reduced by inhibition of α4 and α4ß1 integrins, but not α4ß7 integrins. CONCLUSIONS: Our findings suggest that Teff cell homing to the ileum through the axis α4ß1-VCAM-1 is an essential and nonredundant pathway in CD in vivo, possibly affecting efficacy of clinical treatment with antiadhesion compounds.

Brief Report: A High Rate of ß7+ Gut-Homing Lymphocytes in HIV-Infected Immunological Nonresponders is Associated With Poor CD4 T-Cell Recovery During Suppressive HAART.

OBJECTIVE: Correlation between GALT homing markers on lymphocytes and the low blood CD4 T-cell reconstitution in immunological nonresponders (INRs) has been studied. DESIGN: Thirty-one INRs, 19 immunological responders (IRs), and 12 noninfected controls were enrolled in this study. INRs were defined by an undetectable plasma viral load RNA less than 40 copies per milliliter and CD4 T-cell count <500 cells per cubic milliliter in at least 3 years. METHODS: A complete peripheral and mucosal lymphocyte immunophenotyping was performed on these patients with a focus on the CCR9, CCR6, and α4ß7 gut-homing markers. RESULTS: A highly significant upregulation of α4ß7 on INRs peripheral lymphocytes compared with that of IRs has been observed. This upregulation impacts different lymphocyte subsets namely CD4, CD8, and B lymphocytes. The frequency of ß7 Th17 and Treg cells are increased compared with IRs and healthy controls. The frequency of ß7 CD8 T cells in the blood is negatively correlated with integrated proviral DNA in rectal lymphoid cells in contrast to ß7 CD4 T cells associated with HIV integration. CONCLUSIONS: Alteration of lymphocyte homing abilities would have deleterious effects on GALT reconstitution and could participate to HIV reservoir constitution. These results emphasize the great interest to consider α4ß7-targeted therapy in INR patients to block homing of lymphocytes and/or to directly impair gp120-α4ß7 interactions.

Propionibacterium freudenreichii component 1.4-dihydroxy-2-naphthoic acid (DHNA) attenuates dextran sodium sulphate induced colitis by modulation of bacterial flora and lymphocyte homing.

BACKGROUND AND AIMS: 1.4-Dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator from Propionibacterium freudenreichii, is thought to have a beneficial effect as a prebiotic; however, its in vivo effect on intestinal inflammation remains unknown. The aim of this study was to determine whether oral administration of DHNA can ameliorate dextran sodium sulphate (DSS) induced colitis and to determine the possible underlying mechanisms. METHOD: Colitis was induced in mice by treatment with 2.0% DSS for seven days. DHNA (0.6 or 2.0 mg/kg) was given in drinking water prior to (preventive study) or after (therapeutic study) DSS administration. Colonic damage was histologically scored, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expression and beta7 positive cell infiltration were determined by immunohistochemistry. mRNA levels of proinflammatory cytokines (interleukin (IL)-1beta, IL-6 and tumour necrosis factor alpha (TNF-alpha)) were determined by quantitative real time polymerase chain reaction. In addition, bacterial flora in the caecum, concentrations of short chain acids, and luminal pH were examined. RESULTS: DHNA improved survival rate and histological damage score in mice administered DSS in both the preventive and therapeutic studies. DHNA significantly attenuated the enhanced expression of MAdCAM-1, the increased beta7 positive cell number, and the increased mRNA levels of IL-1beta, IL-6, and TNF-alpha in DSS treated colon. In addition, the decreased number of Lactobacillus and Enterobacteriaceae induced by DSS was recovered by DHNA. Preventive effects on decrease in butyrate concentration and decrease in pH level in mice administered DSS were also observed in the DHNA preventive study. CONCLUSION: DHNA, a novel type of prebiotic, attenuates colonic inflammation not only by balancing intestinal bacterial flora but also by suppressing lymphocyte infiltration through reduction of MAdCAM-1.

Triiodothyronine modulates extracellular matrix-mediated interactions between thymocytes and thymic microenvironmental cells.

OBJECTIVES: Thyroid hormones exert immunomodulatory activities and the thymus is one of their target organs. We previously showed that triiodothyronine (T(3)) modulates thymic hormone production and extracellular matrix (ECM) expression by mouse thymic epithelial cells (TEC). This concept is enlarged herein by studying the effects of T(3) in human TEC preparations including primary cultures derived from thymic nurse cell complexes, as well as human and murine TEC lines. METHODS AND RESULTS: We observed that in all cases, ECM ligands and receptors (such as fibronectin, laminin, VLA-5 and VLA-6) are enhanced in vitro, as ascertained by immunocytochemistry, ELISA and cytofluorometry. Moreover, thymocyte adhesion to these TEC preparations is augmented by T(3). Interestingly, TEC-thymocyte adhesion is also upregulated when thymocytes from T(3)-treated mice adhere to untreated TEC cultures. Such an enhancing effect of T(3) upon TEC-thymocyte interactions is likely due to the increase in the expression of ECM ligands and receptors, since it is prevented when T(3)-treated TEC cultures are incubated with anti-ECM antibodies prior to the adhesion assay. We then tested whether T(3) could modulate interactions between thymocytes and nonepithelial microenvironmental cells, exemplified herein by the phagocytic cells of the mouse thymic reticulum. In fact, in vitro treatment of these cells with T(3) increases ECM ligands and receptors and augments their ability to adhere to thymocytes. Lastly, using immunochemistry-based assays, we showed the presence of the nuclear T(3) receptor in all thymic microenvironmental cell preparations. CONCLUSION: Our data show that T(3) upregulates ECM-mediated heterocellular interactions of thymocytes with distinct thymic microenvironmental cells, in both humans and mice.

Cytokine control of memory B cell homing machinery.

The germinal center (GC) is a pivotal site for the development of B cell memory. Whereas GC B cells do not chemotax to most chemokines and do not express the adhesion receptors L-selectin, alpha(4)beta(7), and cutaneous lymphocyte Ag (CLA), memory B cells respond to various chemotactic signals and express adhesion receptors. In this study, we show that CD40 ligand, IL-2, and IL-10 together drive this transition of GC B cells to memory phenotype in vitro, up-regulating memory B cell markers, chemotactic responses to CXC ligand (CXCL)12, CXCL13, and CCL19, and expression of adhesion receptors L-selectin, alpha(4)beta(7), and CLA. Moreover, addition of IL-4 modulates this transition, preventing chemotactic responses to CXCL12 and CXCL13 (but not to CCL19), and inhibiting the re-expression of L-selectin, but not of CLA or alpha(4)beta(7). CCR7 expression, responsiveness to CCL19, and L-selectin/alpha(4)beta(7) phenotype are coordinately regulated. Thus, IL-2/IL-10 and IL-4 play important and distinctive roles in developing the migratory capacities of memory B cells.

The involvement of lipid rafts in the regulation of integrin function.

Integrin activity on cells such as T lymphocytes is tightly controlled. Here we demonstrate a key role for lipid rafts in regulating integrin function. Without stimulation integrin LFA-1 is excluded from lipid rafts, but following activation LFA-1 is mobilised to the lipid raft compartment. An LFA-1 construct from which the I domain has been deleted mimics activated integrin and is constitutively found in lipid rafts. This correlation between integrin activation and raft localisation extends to a second integrin, alpha4beta1, and the clustering of alpha4beta1 is also raft dependent. Both LFA-1 and alpha4beta1-mediated adhesion is dependent upon intact lipid rafts providing proof of the functional relevance of the lipid raft localisation. Finally we find that non-raft integrins are excluded from the rafts by cytoskeletal constraints. The presence of integrin in lipid rafts under stimulating conditions that activate these receptors strongly indicates that the rafts have a key role in positively regulating integrin activity.

Dual role of H-Ras in regulation of lymphocyte function antigen-1 activity by stromal cell-derived factor-1alpha: implications for leukocyte transmigration.

We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH(2)-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1alpha-induced Akt phosphorylation. SDF-1alpha triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1-mediated transendothelial chemotaxis but not VLA-4-dependent transmigration induced by SDF-1alpha. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1alpha, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1alpha in leukocytes, establishing a complex and bimodal involvement of H-Ras.

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1.

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) and its G-protein-linked receptor CXCR4 are involved in hematopoietic progenitor cell and lymphocyte migration. The integrin VLA-4 is a cell adhesion receptor for CS-1/fibronectin and VCAM-1 and constitutes one of the main adhesion receptors mediating myeloma cell adhesion to bone marrow (BM) stroma in multiple myeloma (MM). It is shown here that MM CD38(hi)CD45RA(-) BM cells and myeloma-derived cell lines expressed CXCR4 and displayed a moderate chemotactic response to SDF-1alpha. Because cell migration in response to SDF-1alpha might require a dynamic regulation of integrin function, it was investigated whether SDF-1alpha can modulate VLA-4 function on myeloma cells. SDF-1alpha rapidly and transiently up-regulated VLA-4-mediated myeloma cell adhesion to both CS-1/fibronectin and VCAM-1, which was inhibited by pertussis toxin and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Modulation of VLA-4-dependent myeloma cell adhesion by SDF-1alpha could contribute to the trafficking and localization of these cells in the BM microenvironment.

VCAM-1-induced inwardly rectifying K(+) current enhances Ca(2+) entry in human THP-1 monocytes.

Hyperpolarization in human leukemia THP-1 monocytes adherent to vascular cell adhesion molecule (VCAM)-1 is due to an induction of inwardly rectifying K(+) currents (I(ir)) (Colden-Stanfield M and Gallin EK, Am J Physiol Cell Physiol 275: C267-C277, 1998). We determined whether the VCAM-1-induced hyperpolarization is sufficient to augment the increase in intracellular free calcium ([Ca(2+)](i)) produced by Ca(2+) store depletion with thapsigargin (TG) and readdition of external CaCl(2) in fura 2-loaded THP-1 monocytes. Whereas there was a 2.1-fold increase in [Ca(2+)](i) in monocytes bound to glass for 5 h in response to TG and CaCl(2) addition, adherence to VCAM-1 produced a 5-fold increase in [Ca(2+)](i). Depolarization of monocytes adherent to VCAM-1 by I(ir) blockade or exposure to high [K(+)] abolished the enhancement of the peak [Ca(2+)](i) response. In monocytes bound to glass, hyperpolarization of the membrane potential with valinomycin, a K(+) ionophore, to the level of hyperpolarization seen in cells adherent to VCAM-1 produced similar changes in peak [Ca(2+)](i). Adherence of monocytes to E-selectin produced a similar peak [Ca(2+)](i) to cells bound to glass. Thus monocyte adherence to the physiological substrate VCAM-1 produces a hyperpolarization that is sufficient to enhance Ca(2+) entry and may impact Ca(2+)-dependent monocyte function.

Angiotensin II (AT(1)) receptor blockade reduces vascular tissue factor in angiotensin II-induced cardiac vasculopathy.

Tissue factor (TF), a main initiator of clotting, is up-regulated in vasculopathy. We tested the hypothesis that chronic in vivo angiotensin (ANG) II receptor AT(1) receptor blockade inhibits TF expression in a model of ANG II-induced cardiac vasculopathy. Furthermore, we explored the mechanisms by examining transcription factor activation and analyzing the TF promoter. Untreated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) feature hypertension and severe left ventricular hypertrophy with focal areas of necrosis, and die at age 7 weeks. Plasma and cardiac ANG II was three- to fivefold increased compared to Sprague-Dawley rats. Chronic treatment with valsartan normalized blood pressure and coronary resistance completely, and ameliorated cardiac hypertrophy (P < 0.001). Valsartan prevented monocyte/macrophage infiltration, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activation, and c-fos expression in dTGR hearts. NF-kappaB subunit p65 and TF expression was increased in the endothelium and media of cardiac vessels and markedly reduced by valsartan treatment. To analyze the mechanism of TF transcription, we then transfected human coronary artery smooth muscle cells and Chinese hamster ovary cells overexpressing the AT(1) receptor with plasmids containing the human TF promoter and the luciferase reporter gene. ANG II induced the full-length TF promoter in both transfected cell lines. TF transcription was abolished by AT(1) receptor blockade. Deletion of both AP-1 and NF-kappaB sites reduced ANG II-induced TF gene transcription completely, whereas the deletion of AP-1 sites reduced transcription. Thus, the present study clearly shows an aberrant TF expression in the endothelium and media in rats with ANG II-induced vasculopathy. The beneficial effects of AT(1) receptor blockade in this model are mediated via the inhibition of NF-kappaB and AP-1 activation, thereby preventing TF expression, cardiac vasculopathy, and microinfarctions.

Survival signals within the tumour microenvironment suppress drug-induced apoptosis: lessons learned from B lymphomas.

The suppression of apoptosis is one mechanism by which tumours become drug resitant. Extracellular signals from the germinal centre (GC) of secondary lymphoid tissue can rescue B cells from physiological- and chemotherapy-induced apoptosis. Such survival signals include CD40 receptor ligation, interleukin-4 (IL-4) receptor stimulation and the interaction of the integrin ligand VCAM-1 with its receptor. The GC environment was modelled in vitro by providing B lymphoma cells with these survival signals. JLP119 B lymphoma cells underwent apoptosis after exposure to the topisomerase II inhibitor etoposide and this was dramatically reduced when the cells were cultured in the GC system. CD40 receptor ligation resulted in increased levels of Bcl-XL. Etoposide diminished the binding between Bax and Bcl-XL but this was restored by IL-4 and VCAM-1 triggered signals. These data demonstrate combined effects of three microenvironmental signals on the Bcl-2 family and illustrate the potential importance of such signalling pathways in drug resistance of tumour cells.

The disintegrin eristostatin interferes with integrin alpha 4 beta 1 function and with experimental metastasis of human melanoma cells.

Peptides containing the integrin recognition sequence, RGD, can inhibit experimental metastasis of mouse melanoma cells, but the integrin(s) affected in these experiments is unknown. Besides "classical" RGD-binding integrins such as alpha 5 beta 1 and alpha v beta 3, RGD has been reported to bind alpha 4 beta 1, and mAbs to alpha 4 beta 1 can inhibit melanoma metastasis. We investigated the mode of action of the disintegrin eristostatin, an RGD-containing peptide isolated from snake venom, in a human melanoma experimental metastasis model. Lung colonization following i.v. injection of MV3 cells in nude mice was strongly inhibited by eristostatin. MV3 cells bound FITC-eristostatin and adhered to eristostatin-coated wells. This adhesion was partially inhibited by a GRGDSP peptide and by alpha 4 mAb. Binding of FITC-eristostatin to Jurkat cells and adhesion of Jurkat (but not K562) cells to eristostatin-coated wells further suggested that eristostatin binds alpha 4 beta 1, even though, again, alpha 4 mAb only partially inhibited adhesion. Expression of alpha 4 beta 1 was enhanced in metastatic melanoma cells compared to normal melanocytes and nonmetastatic melanoma cells. Finally, eristostatin inhibited adhesion of both MV3 and CHO alpha 4 cells to the alpha 4 beta 1-ligand VCAM-1, while adhesion to other ligands via other integrins was not affected. These findings demonstrate that inhibition of melanoma cell metastasis by RGD-containing peptides such as eristostatin, may be due to interference with alpha 4 beta 1-VCAM binding, in addition to inhibition of the classical RGD-binding integrins.

IL-7 activates alpha4beta1 integrin in murine thymocytes.

IL-7, a cytokine produced by thymic epithelium, was shown to induce adhesion of murine thymocytes to gelatin-coated membranes. A major binding component of gelatin was identified as fibronectin. IL-7-induced adhesion was observed for all of the major thymocyte subsets, including double-negative, double-positive, and single-positive cells, and specific IL-7R were verified on each subset. Fibronectin binding was mediated via alpha4beta1 integrin (VLA-4), which is expressed at high levels on thymocytes. VLA-4 surface expression was not increased following IL-7 treatment, but was shown to undergo rapid tyrosine phosphorylation on the beta1 subunit. This tyrosine phosphorylation was blocked by genistein, which also blocked IL-7-induced adhesion. IL-7 was detected on the extracellular matrix of the thymus, suggesting that it could promote matrix association through an integrin pathway.

Differential modulation of adhesion markers with ex vivo expansion of human umbilical CD34+ progenitor cells.

The effect of different expansion protocols on the expression levels of CD49dw/CD29 (VLA-4), CD11a/CD18 (LFA-1), CD31 (PECAM-1), CD44, and CD34 was determined after cord blood CD34+ cells were cultured for defined periods with the following: 1) A growth factor mix (GFmix) containing interleukin (IL)-1, IL-3, IL-6, kit ligand (KL), G-CSF, GM-CSF, and erythropoietin (Epo); 2) IL-3 + KL; and 3) HS-5 (a human stromal cell line supernatant) + KL. Before culturing, cord blood CD34+ cells (> 95% purity) were 94 +/- 5% CD31+, 98 +/- 1% CD44+, 66 +/- 29% VLA-4+, and 68 +/- 18% LFA-1+ (mean +/- SEM). Immunophenotyping and morphological examination of pre- and post-cultured cells indicated that GFmix preferentially supported erythroid development, while IL-3+KL and HS-5+KL preferentially supported myeloid development. Similar to what other investigators have reported, there was an absolute increase in CD34+ cell numbers as well as clonogenic precursors with ex vivo expansion. However, the majority of clonogenic precursors post-expansion expressed CD34 antigen at reduced levels. Examination of adhesion molecules indicated that a majority of cells cultured with GFmix expressed PECAM-1 and LFA-1 at undetectable levels, but PECAM-1 and LFA-1 levels remained essentially unchanged when cells were cultured with IL-3+KL and HS-5+KL. Overall VLA-4 expression levels slightly increased and CD44 expression levels were more heterogeneous with ex vivo expansion. Nevertheless, LFA-1, VLA-4, PECAM-1, and CD44 expression levels remained essentially unchanged on cultured progeny retaining a CD34 phenotype, independent of the culture system used. Together these results indicate that differential modulation of adhesion markers occur with different culture conditions, yet adhesion receptor expression levels on progeny cells retaining a CD34 phenotype are essentially maintained independent of the culture conditions. And although there is an absolute increase in CD34+ cells after ex vivo expansion, a majority of clonogenic precursors have reduced levels of CD34 antigen.

Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells.

Rotavirus contains two outer capsid viral proteins, the spike protein VP4 and major capsid component VP7, both of which are implicated in cell entry. We show that VP4 and VP7 contain tripeptide sequences previously shown to act as recognition sites for integrins in extracellular matrix proteins. VP4 contains the alpha2beta1 integrin ligand site DGE. In VP7, the alphaxbeta2 integrin ligand site GPR and the alpha4beta1 integrin ligand site LDV are embedded in a novel disintegrin-like domain that also shows sequence similarity to fibronectin and the tie receptor tyrosine kinase. Microorganism sequence homology to these ligand motifs and to disintegrins has not been reported previously. In our experiments, peptides including these rotaviral tripeptides and mAbs directed to these integrins specifically blocked rotavirus infection of cells shown to express alpha2beta1 and beta2 integrins. Rotavirus VP4-mediated cell entry may involve the alpha2beta1 integrin, whereas VP7 appears to interact with alphaxbeta2 and alpha4beta1 integrins.

Granulocyte-macrophage colony-stimulating factor regulates the functional adhesive state of very late antigen-4 expressed by eosinophils.

As very late antigen-4 (VLA-4) can exist in different functional states, we have sought to determine whether a cytokine expressed by inflamed endothelium (i.e., granulocyte-macrophage CSF (GM-CSF)) could regulate the functional state of VLA-4 expressed by eosinophils. Using a micropipette single cell adhesion assay able to measure the strength of adhesion forces, eosinophils exhibited low levels of basal adhesion to unstimulated endothelium (separation force, 0.022 +/- 0.003 mdynes). In contrast, individual eosinophils bound to IL-1beta-stimulated endothelium (0.49 +/- 0.02 mdynes), TNF-stimulated endothelium (0.62 +/- 0.05 mdynes), or IL-4-stimulated endothelium (0.11 +/- 0.01 mdynes) with increased avidity as assessed by separation force. Eosinophil binding to IL-4-stimulated endothelium was significantly inhibited by neutralizing Abs to either vascular cell adhesion molecule (VCAM) or VLA-4. The strength of eosinophil adhesion to VCAM (0.31 +/- 0.02 mdynes) or to connecting segment-1 (CS-1) (0.18 mdynes) was greater than the strength of eosinophil adhesion to unstimulated endothelium (0.02 mdynes), but was less than the strength of eosinophil adhesion to IL-1beta-stimulated endothelium (0.49 +/- 0.02 mdynes). After incubating eosinophils for 30 min with GM-CSF, the mean adhesion strength of eosinophils to CS-1 and VCAM increased significantly by 84 and 54%, respectively, compared with that of controls. This increased binding of eosinophils to VCAM or CS-1 was not due to alterations in VLA-4 receptor number (assessed by FACS analysis) or alterations in VLA-4 receptor distribution (assessed by confocal microscopy). These studies suggest that endothelial-derived cytokines such as GM-CSF have the potential to alter the functional state of eosinophil-expressed VLA-4 from a low affinity state to a high affinity state.

Are murine marginal-zone macrophages the splenic white pulp analog of high endothelial venules?

The entry of lymphocytes into the spleen, in contrast to lymph nodes, does not involve high endothelial venule (HEV) interaction. The precise point of entry, as well as the mechanism by which lymphocytes enter the lymphoid areas of the spleen, remains controversial. We examined in detail the effect of two agents, pertussis toxin (PT) and the sulfated polysaccharide fucoidan, on splenic lymphocyte entry and positioning. These have previously been shown to interfere with lymphocyte extravasation across HEV. PT prevents lymphocyte extravasation, but not binding, to HEV, whereas fucoidan prevents binding and thus subsequent extravasation. Studies presented here show that pretreatment of murine lymphocytes with PT does not numerically affect entry into spleen, but profoundly alters lymphocyte positioning within the spleen. When fluorescently labeled, PT-treated lymphocytes are injected intravenously, they initially accumulate in the marginal zone, in apparent association with the layer of marginal zone macrophages (MZM phi) which form a shell around the white pulp. They fail to traverse this layer into the white pulp, and subsequently localize in the red pulp. In contrast, untreated cells initially appear in the marginal zone, then continue to migrate into the white pulp after traversing the MZM phi layer. The localization of PT-pretreated lymphocytes adjacent to the MZM phi layer is disrupted by intravenous administration of fucoidan. Using a flow cytometric assay of aggregation between MZM phi and lymphocytes, we confirmed that fucoidan is also able to inhibit this association in vitro, whereas PT has no effect on this interaction. We propose that MZM phi in the mouse are the splenic analog of HEV, forming the port of entry of lymphocytes into the white pulp of the spleen.

Differential effects of dexamethasone on the thymus and spleen: alterations in programmed cell death, lymphocyte subsets and activation of T cells.

The kinetics of DEX-induced changes in lymphocytes from the thymus and spleen of normal mice were examined in relation to cell numbers, programmed cell death (PCD), in vitro proliferative response to anti-CD3 antibodies or Con-A, and changes in lymphocyte subsets by flow cytometry. The above aspects were examined at 48, 24, 18, and 3 h after a single intraperitoneal injection of DEX. Profound reduction of thymocyte numbers was noticed particularly at 48 (74-84%) and 24 (43-55%) h after DEX administration. PCD of thymocytes was not detectable at 48 h, marginally detectable at 24 h, markedly evident at 18 h, and minimally detectable at 3 h pi of DEX. PCD in splenic lymphocytes of DEX-treated mice was not clearly evident at these time points. Thymocytes from mice exposed to DEX (48 or 24 h) proliferated vigorously when cultured in the presence of anti-CD3 or Con-A, thereby suggesting that the remaining thymocytes can transduce activation signals. In contrast, splenic lymphocytes from the same animals responded poorly to these stimulants. Flow cytometric studies revealed that there was a marked increase in number of thymocytes expressing CD3+ (4-6 fold) and alpha beta TCR+ (2-7 fold) surface molecules. On the other hand, no major changes in CD3+ or alpha beta TCR+ cells were noticed in the spleen of DEX-treated mice. Although the total numbers of cells expressing heat stable antigen, M1/69, were not markedly altered after DEX treatment, the fluorescent intensity profile was modified. There were no remarkable changes in CD45RB+ cells in these mice. CD44+ cells were not decreased in DEX-treated thymocytes or splenic lymphocytes. Our results suggest that DEX has differential effects on the thymus and the spleen.

TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan.

TGF-beta is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-beta-stimulated locomotion and show that TGF-beta triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-beta 1 and by stimulating the production of bioactive TGF-beta 1 in tumor cells transfected with TGF-beta 1 under the control of the metallothionein promoter. TGF-beta 1-induced locomotion was suppressed by antibodies that prevented HA/RHAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-beta 1-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-beta with panspecific TGF-beta antibodies and by inhibition of TGF-beta 1 secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-beta, and the pathway by which TGF-beta stimulates locomotion uses the HA receptor RHAMM and HA.