Expression Of LFA-1 Research Articles

Both Regulatory T Cells and Antitumor Effector T Cells Are Primed in the Same Draining Lymph Nodes during Tumor Progression

The peripheral tolerance mechanism prevents effective antitumor immunity, even though tumor cells possess recognizable tumor-associated Ags. Recently, it has been elucidated that regulatory T cells (Treg) play a critical role in maintaining not only self-tolerance, but also tolerance of tumor cells. However, because the Treg that maintain self-tolerance arise naturally in the thymus and are thought to be anergic in peripheral, it is still unclear where and when Treg for tumor cells are generated. In this study we analyze tumor-draining lymph nodes (LNs) and demonstrate that both antitumor effector T cells and Treg capable of abrogating the antitumor reactivity of the effector T cells are primed in the same LNs during tumor progression. The regulatory activity generated in tumor-draining LNs exclusively belonged to the CD4(+) T cell subpopulation that expresses both CD25 and a high level of CD62L. Forkhead/winged helix transcription factor gene expression was detected only in the CD62L(high)CD4(+)CD25(+) T cells. CD62L(high)CD4(+)CD25(+) Treg and CD62L(low)CD4(+)CD25(+) T cells, which possess effector T cell functions, had comparable expression of LFA-1, VLA-4, CTLA-4, lymphocyte activation gene-3, and glucocorticoid-induced TNFR. Thus, only CD62L expression could distinguish regulatory CD4(+)CD25(+) cells from effector CD4(+)CD25(+) cells in draining LNs as a surface marker. The Treg generated in tumor-draining LNs possess the same functional properties as the Treg that arise naturally in the thymus but recognize tumor-associated Ag. CD62L(high)CD4(+)CD25(+) Treg contained a subpopulation that expressed CD86. Blocking experiments revealed that ligation of CTLA-4 on effector T cells by CD86 on Treg plays a pivotal role in regulating CD4(+) effector T cells.

The cachectic mediator proteolysis inducing factor activates NF-κB and STAT3 in human Kupffer cells and monocytes

A novel proteoglycan, proteolysis inducing factor (PIF), is capable of inducing muscle proteolysis during the process of cancer cachexia, and of inducing an acute phase response in human hepatocytes. We investigated whether PIF is able to activate pro-inflammatory pathways in human Kupffer cells, the resident macrophages of the liver, and in monocytes, resulting in the production of pro-inflammatory cytokines. Normal liver tissue was obtained from patients undergoing partial hepatectomy and Kupffer cells were isolated. Monocytes were isolated from peripheral blood. Following exposure to native PIF, pro-inflammatory cytokine production from Kupffer cells and monocytes was measured and the NF-kappaB and STAT3 transcriptional pathways were investigated using electrophoretic mobility shift assays. We demonstrate that PIF is able to activate the transcription factor NF-kappaB and NF-kappaB-inducible genes in human Kupffer cells, and in monocytes, resulting in the production of pro-inflammatory cytokines such as TNF-alpha, IL-8 and IL-6. PIF enhances the expression of the cell surface molecules LFA-1 and CD14 on macrophages. PIF also activates the transcription factor STAT3 in Kupffer cells. The pro-inflammatory effects of PIF, mediated via NF-kappaB and STAT3, are important in macrophage behaviour and may contribute to the inflammatory pro-cachectic process in the liver.

Interaction between human lung fibroblasts and T-lymphocytes prevents activation of CD4+ cells

BackgroundT lymphocytes are demonstrated to play an important role in several chronic pulmonary inflammatory diseases. In this study we provide evidence that human lung fibroblasts are capable of mutually interacting with T-lymphocytes leading to functionally significant responses by T-cells and fibroblasts.MethodsHuman lung fibroblast were co-cultured with PMA-ionomycin activated T-CD4 lymphocytes for 36 hours. Surface as well as intracellular proteins expression, relevant to fibroblasts and lymphocytes activation, were evaluated by means of flow cytometry and RT-PCR. Proliferative responses of T lymphocytes to concanavalin A were evaluated by the MTT assay.ResultsIn lung fibroblasts, activated lymphocytes promote an increase of expression of cyclooxygenase-2 and ICAM-1, expressed as mean fluorescence intensity (MFI), from 5.4 ± 0.9 and 0.7 ± 0.15 to 9.1 ± 1.5 and 38.6 ± 7.8, respectively. Fibroblasts, in turn, induce a significant reduction of transcription and protein expression of CD69, LFA-1 and CD28 in activated lymphocytes and CD3 in resting lymphocytes. In activated T lymphocytes, LFA-1, CD28 and CD69 expression was 16.6 ± 0.7, 18.9 ± 1.9 and 6.6 ± 1.3, respectively, and was significantly reduced by fibroblasts to 9.4 ± 0.7, 9.4 ± 1.4 and 3.5 ± 1.0. CD3 expression in resting lymphocytes was 11.9 ± 1.4 and was significantly reduced by fibroblasts to 6.4 ± 1.1. Intracellular cytokines, TNF-alpha and IL-10, were evaluated in T lymphocytes. Co-incubation with fibroblasts reduced the number of TNF-alpha positive lymphocytes from 54,4% ± 6.12 to 30.8 ± 2.8, while IL-10 positive cells were unaffected. Finally, co-culture with fibroblasts significantly reduced Con A proliferative response of T lymphocytes, measured as MTT absorbance, from 0.24 ± 0.02 nm to 0.16 ± 0.02 nm. Interestingly, while the activation of fibroblasts is mediated by a soluble factor, a cognate interaction ICAM-1 mediated was demonstrated to be responsible for the modulation of LFA-1, CD28 and CD69.ConclusionFindings from this study suggest that fibroblasts play a role in the local regulation of the immune response, being able to modulate effector functions of cells recruited into sites of inflammation.

Spermine, a Natural Polyamine, Suppresses LFA-1 Expression on Human Lymphocyte

Natural polyamines, spermine, spermidine, and putrescine, play a pivotal role in the regulation of gene expression; therefore, the age-dependent decreases and the disease-dependent increases in polyamine synthesis suggest a possible contribution of polyamines to the age-related and disease-associated changes in cellular function. In this study, we examined the effects of polyamines on the cellular function and the expression of adhesion molecules on human PBMCs from healthy volunteers. Flow cytometry revealed that PBMCs cultured with spermine decreased mean fluorescent intensities (MFIs) of CD11a and CD18 in the lymphocyte light-scattered region, but not in the monocyte region. This suppression was observed in a dose- and time-dependent manner and found nonspecifically on all cell subsets we tested (CD3(+), CD4(+), CD8(+), CD19(+), CD45RA(+), CD45RO(+), CD4(+)CD45RA(+), CD4(+)CD45RO(+), CD8(+)CD45RA(+), CD8(+)CD45RO(+)). The decreases of CD11a and CD18 MFIs were accompanied by the decrease in adherent capacity of PBMCs to HUVECs. Spermine did not hinder cell activities or cell viability. Among 42 healthy volunteers (mean, 49.5 years old; from 26 to 69), blood spermine levels inversely correlated with the CD11a MFIs of cells in the lymphocyte region (r = -0.48; p = 0.001), but not with those in the monocyte region. The effects of spermidine seemed weaker than those of spermine, and blood spermidine levels had no correlation with CD11a MFIs of the lymphocyte region. Putrescine had no effect on the expressions of membrane molecules. Polyamines, especially spermine, decrease LFA-1 (CD11a/CD18) expression on human lymphocyte and adhesion capacity of PBMCs to HUVECs.

Differential expression of LFA-3, Fas and MHC Class I on Ad5- and Ad12-transformed human cells and their susceptibility to lymphokine-activated killer (LAK) cells

Adenovirus (Ad) E1A is a potent oncogene and has been shown to deregulate the expression of a large number of cellular genes leading to cellular transformation. Here we have analysed the expression of several immunomodulatory molecules on the surface of a set of human cell lines transformed with either Ad12 or Ad5. Human cells transformed with Ad12 demonstrated reduced expression of cell surface LFA-3, Fas and MHC class I when compared to Ad5-transformed cells. Furthermore, Ad12-transformed human cell lines demonstrated greater susceptibility to lysis by lymphokine-activated killer (LAK) cells, compared to Ad5-transformed human cell lines. In contrast, previous studies with rodent cells showed that both Ad5- and Ad12-transformed rat cells were susceptible to LAK cells. Thus, transformation of human cells with Ad5 or Ad12 results in differences in the expression of immunomodulatory molecules on the cell surface and differential recognition of these virus-transformed cells by immune effector cells.

Recombinant expression of bovine LFA-1 and characterization of its role as a receptor for Mannheimia haemolytica leukotoxin

Mannheimia ( Pasteurella) haemolytica leukotoxin (LktA) is the primary virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM), a disease which causes major economic loss to the US cattle industry annually. Recent work from our laboratory using an antibody-based approach has shown that LktA binds to bovine LFA-1 in target cells. While this study suggests that LFA-1 might be a specific receptor, it remains to be conclusively shown that LFA-1 is sufficient to induce susceptibility to LktA. It was of interest to determine if functionally active bovine LFA-1 could be reconstituted on a LFA-1 negative cell line and reconstitute susceptibility to LktA. Here, we report the successful recombinant expression of bovine LFA-1 on the cell surface of the human erythroleukemic K562 cell line. The BoLFA-1 transductant expresses bovine CD18 and CD11a as a heterodimer. We found that LktA binds to both the CD18 and CD11a subunits of BoLFA-1 cells. Exposure of BoLFA-1 cells to LktA, induced tyrosine phosphorylation of the CD18 tail, elevation of intracellular calcium, and cytolysis. This is the first report on recombinant expression of functionally active bovine LFA-1 by transduction into an LktA-non-susceptible human cell line.

Naive, Effector, and Memory T Lymphocytes Efficiently Scan Dendritic Cells In Vivo: Contact Frequency in T Cell Zones of Secondary Lymphoid Organs Does Not Depend on LFA-1 Expression and Facilitates Survival of Effector T Cells

Contact between T cells and dendritic cells (DCs) is required for their subsequent interaction leading to the induction of adaptive immune responses. Quantitative data regarding the contact frequencies of T cell subsets in different lymphoid organs and species are lacking. Therefore, naive, effector, and memory CD4 T cells were injected into rats in absence of the cognate Ag, and 0.5-96 h later, spleen, lymph nodes, and Peyer's patches were removed. Cryosections were analyzed for contact between donor T cells and endogenous DCs in the T cell zone, and donor cell proliferation. More than 60% of injected naive CD4 T cells were in contact with endogenous DCs at all time points and in all organs analyzed. Surprisingly, we were unable to detect any differences between naive, effector, and memory CD4 T cells despite different expression levels of surface molecules. In addition, contact frequency was similar for T cells in lymphoid organs of rats, mice, and humans; it was unaffected by the absence of LFA-1 (CD11a/CD18), and sustained effector T cells in an activated state. Thus, the architecture of the T cell zone rather than expression patterns of surface molecules determines the contact efficiency between T cells and DCs in vivo.

Expression of adhesion molecules and RANTES in kidney transplant from nonheart-beating donors

The main difference between cadaveric kidneys from donors with a heartbeat (HBD) and kidneys from nonheart-beating donors (NHBD) is related to warm ischemia/reperfusion time which constitutes an acute inflammatory process. On the contrary, brain death induces in HBD expression of pro-inflammatory adhesion molecules, making it important to evaluate this kind of molecules in both types of donors. Human renal biopsies from NHBD, HBD and normal kidneys (ischemia time = 0) were taken and frozen just before transplant. A semi-quantitative RT-PCR method was used to determine intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), lymphocyte function associated antigen (LFA-1), LFA-3, CD40, CD40 ligand (CD40L) and RANTES (regulated upon activation, normal T-cell expressed and secreted) gene expression. We have detected an elevated relative gene expression of ICAM-1, VCAM-1 and RANTES in NHBD biopsies compared with normal kidneys. In the case of RANTES, the gene expression from NHBD biopsies was higher than observed in HBD biopsies. The rest of genes were not augmented in any group. Preliminary data about early outcome of transplants indicates a correlation between pretransplant RANTES high gene expression levels and early post-transplant acute rejection. The gene expression of pro-inflammatory molecules like adhesion molecules and RANTES is augmented in kidneys from cadaveric NBD just before transplant. The expression is higher probably because of the prolonged warm ischemia period. A larger clinical study is necessary to clarify the effects of these variable expressions on the transplant outcome.

Exercise paradoxically modulates oxidized low density lipoproteininduced adhesion molecules expression and trans-endothelial migration of monocyte in men

Physical exercise can affect the risk of cardiovascular disease. Oxidized-low density lipoprotein (ox-LDL) promotes transendothelial migration (TEM) of monocyte, thereby accelerating the pathogenesis of atherosclerosis. This study investigated how exercise intensity affects monocyte/EC interactions under ox-LDL-mediated condition. Light- (LIE), moderate- (MIE) and high- (HIE) intensity exercise (i.e., 40%, 60%, and 80% VO2max, respectively) on a bicycle ergometer in 18 sedentary healthy men were performed on three separate occasions. Before and immediately after exercise, ox-LDL-promoted expressions of monocyte adhesion molecules and TEM of monocyte, as well as oxidation of LDL and amounts of soluble adhesion molecules in plasma were measured. Analytical results showed that (1) ox-LDL furthered monocyte L-selectin shedding and Mac-1 expression, and an attendant increase in TEM of monocyte, while treating the monocyte with Mac-1 antibody inhibited the ox-LDL-promoted TEM of monocyte; (2) under ox-LDL-treated condition, MIE increased monocyte Mac-1 and LFA-1 expressions, enhancing the TEM of monocyte, whereas HIE downregulated monocyte Mac-1 expression, suppressing the TEM of monocyte; (3) LIE decreased basal LFA-1 expression as well as basal and ox-LDL-promoted TEM of monocyte; and (4) MIE and HIE, but not LIE, elevated plasma ox-LDL level, while there were no significant changes in sL-selectin, sE-selectin, sICAM-1, and sVCAM-1 following these exercises. Therefore, we conclude that monocyte activation and subsequent TEM promoted by ox-LDL are changed by short-term exercise in an intensity-dependent manner. These findings provide a new insight into the may aid the development of suitable exercise intensity enable people to prevent early atherogenesis.

Dietary supplementation with Cyperus esculentus L (tiger nut) tubers attenuated atherosclerotic lesion in apolipoprotein E knockout mouse associated with inhibition of inflammatory cell responses

Atherosclerosis is a pathological process, where recruitment of mononuclear cells results in the growth of fibro-fatty plaques. Apolipoprotein E (ApoE) is a legend for the uptake of lipoproteins, and deficiency of ApoE leads to the accumulation of low-density lipoprotein. Utilizing ApoE-/- mouse, which spontaneously develop atherosclerosis on low-fat chow diets, the present study showed that feeding these mice on a diet supplemented with the whole tubers of Cyperus esculentus L (tiger nut) resulted in attenuation of the development of atherosclerotic lesions. The anti-atherosclerotic effect was associated with a decrease in the number of monocytes, macrophages and dendritic cells in blood, and the expression of IL-2Rα and LFA-1 by these cells. Further, in vitro proliferation of blood and spleen cells from tiger nut-fed ApoE-/- mice showed lower proliferation in responses to ConA and LPS, a T and B cell mitogen, respectively. Further, in vitro treatment of blood and spleen cells with water or ethanol extracts of tiger nut markedly increased their proliferation in response to ConA. Collectively, these data indicate that ingredients of tiger nut tubers exhibit anti-inflammatory properties upon inflammation, and immunostimulatory effects in immunocompetent hosts.

Complexities of the pathogenesis ofMannheimia haemolyticaandHaemophilus somnusinfections: challenges and potential opportunities for prevention?

Progress in producing improved vaccines against bacterial diseases of cattle is limited by an incomplete understanding of the pathogenesis of these agents. Our group has been involved in investigations of two members of the family Pasteurellaceae, Mannheimia haemolytica and Haemophilus somnus, which illustrate some of the complexities that must be confronted. Susceptibility to M. haemolytica is greatly increased during active viral respiratory infection, resulting in rapid onset of a severe and even lethal pleuropneumonia. Despite years of investigation, understanding of the mechanisms underlying this viral-bacterial synergism is incomplete. We have investigated the hypothesis that active viral infection increases the susceptibility of bovine leukocytes to the M. haemolytica leukotoxin by increasing the expression of or activating the beta2 integrin CD11a/CD18 (LFA-1) on the leukocyte surface. In vitro exposure to proinflammatory cytokines (i.e. interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) increases LFA-1 expression on bovine leukocytes, which in turn correlates with increased binding and responsiveness to the leukotoxin. Alveolar macrophages and peripheral blood leukocytes from cattle with active bovine herpesvirus-1 (BVH-1) infection are more susceptible to the lethal effects of the leukotoxin ex vivo than leukocytes from uninfected cattle. Likewise, in vitro incubation of bovine leukocytes with bovine herpesvirus 1 (BHV-1) potentiates LFA-1 expression and makes the cells more responsive to leukotoxin. A striking characteristic of H. somnus infection is its propensity to cause vasculitis. We have shown that H. somnus and its lipo-oligosaccharide (LOS) trigger caspase activation and apoptosis in bovine endothelial cells in vitro. This effect is associated with the production of reactive oxygen and nitrogen intermediates, and is amplified in the presence of platelets. The adverse effects of H. somnus LOS are mediated in part by activation of endothelial cell purinergic receptors such as P2X7. Further dissection of the pathways that lead to endothelial cell damage in response to H. somnus might help in the development of new preventive or therapeutic regimens. A more thorough understanding of M. haemolytica and H. somnus virulence factors and their interactions with the host might identify new targets for prevention of bovine respiratory disease.

The Expression of the CD21 Antigen in Non-Hodgkin's Lymphoma Is Involved in LFA-1 Expression and Tumor Survival.

CD21/complement receptor type 2 is a membrane protein expressed on B lymphocytes, follicular dendritic cells, early thymocytes, and a subset of mature T lymphocytes. This major B cell antigen appears later than CD19 and CD20 in cell differentiation and is lost from the cell surface during the early stages of B cell activation. CD21 is characterized not only as a complement ligand but also as an adhesion molecule of the cell surface that is dependent on CD23 expression, suggesting that it may be involved in lymphocyte trafficking and several immune responses. Recently two groups have reported that CD21 expression is significantly negatively associated with mortality in diffuse large B cell lymphomas. From these reports and clinical observations, we have transfected the CD21 gene into a CD21/CD23-negative lymphoma cell line (Namalwa). Interestingly, all established CD21+ transfectants (six clones from different bulks) showed homotypic aggregation during in vitro cell culturing, and none of the anti-CD21 antibodies prevented this cell aggregation. Moreover, these CD21+ transfectants were easily rejected in vivo when they were injected in nude mice compared to vector-only transfectants and parental cells as controls. These observations suggest that other adhesion molecules may be involved in CD21-induced cell aggregation and reduced tumorgenesis in vivo. After the screening of several integrins, CD21+ transfectants highly expressed LFA-1 (CD11a/CD18) on their cell surfaces compared to vector-only transfectants and parental cells. CD21+ transfectants displayed tight adhesion when in contact with recombinant human ICAM-1, and cell aggregation of CD21+ transfectants was blocked by an anti-LFA-1 monoclonal antibody. Northern and Western analysis and confocal laser scanning microscopic analysis suggested that the CD21 expression made the transfer of LFA-1 from the cytoplasm to the cell surface. We conclude that the expression of the CD21 molecule is strongly associated with the LFA-1 expression on the cell surface, independent of the CD23 molecule, and this phenomenon may be involved in tumor survival in non-Hodgkin's lymphoma in vivo.

Higher Expression Levels of the Adhesion Molecules VLA-4 and ICAM-1 on Multiple Myeloma Cells after Chemotherapy Are Associated with Survival of a Multidrug Resistant Subpopulation in Vivo.

Objectives: Primary drug resistance is a major problem in multiple myeloma (MM), an incurable disease of the bone marrow. Adhesion of multiple myeloma cells to bone marrow stromal cells (BMSC) has been shown to cause strong primary resistance. The adhesion molecules LFA-1 and VLA-4 are upregulated upon treatment with cytotoxic agents. Furthermore, we have shown that the corresponding ligands on HS-5 BMSCs, VCAM-1 and ICAM-1, are upregulated after incubation with melphalan, suggesting increase of adhesion mediated drug resistance after chemotherapy. In this context, the expression levels of important adhesion molecules on MM cells of consecutive MM patients before and after chemotherapy have been determined in this study.Methods: The expression levels of VLA-1, VLA-4, VLA-5, LFA-1, VCAM, ICAM-1, CD138, CD38, and CD56 were determined on MM cell lines, HS-5 stromal cells, and primary myeloma cells of 20 consecutive patients by flow cytometry in comparison to isotype control. 9 patients had been pre-treated (mostly induction chemotherapy and high dose melphalan with stem cell rescue) and 11 patients had been at diagnosis without treatment. Interpatient comparison of treated and untreated patients was performed. Intrapatient analysis (before and after high dose chemotherapy) will be performed in the follow up.Results: VLA-4 and ICAM-1 are upregulated after chemotherapy by 54% and 64%, respectively. Similar upregulation of CD38 could be observed (62%), whereas CD138 shows downregulation by about 50%. CD56, VCAM, and LFA-1 expression was not significantly altered after chemotherapy.Conclusion: The adhesion molecules VLA-4 and ICAM-1, which are essential for MM-BMSC interaction, are upregulated after chemotherapy. This finding supports our preclinical data and the hypothesis, that adhered, primary drug resistant MM cells are selected by chemotherapy and herewith contribute to multidrug resistance in multiple myeloma.

Modification of B-CLL Cells Via Infection with a Replication-Defective MVA Virus Encoding Three Costimulatory Molecules: A Potential Approach to Tumor Cell Immunotherapy of B-CLL.

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a lymphoproliferative disorder characterized by the accumulation of slowly dividing, apoptosis-defective, clonal CD5+ B cells that typically involve the bone marrow, lymph nodes, spleen and blood of affected individuals. New therapeutic approaches are necessary to be investigated for the treatment of B-CLL. Several characteristics of the neoplastic B cells make B-CLL a good candidate for immunotherapy; evidence exists that B-CLL cells can be recognized by autologous anti-tumor T cells in an MHC-restricted manner. B-CLL cells, however, are themselves ineffective antigen-presenting cells mainly because of their inadequate costimulatory capacity. We have investigated the ability of in vitro manipulated B-CLL cells that express a triad of costimulatory molecules, designated TRICOM, to stimulate effective anti-tumor T-cell responses and their potential as cancer vaccines. Delivery of transgenes to the B-CLL cells could be accomplished through the use of a modified vaccinia virus strain Ankara designated MVA. This is a highly attenuated, non-replicative virus that has been demonstrated to be safe and well tolerated in both animals and humans. Here we report the use of a recombinant MVA vector to deliver the simultaneous expression of three human costimulatory molecules (B7-1, ICAM-1, LFA-3) on the surface of B-CLL cells. Infection was conducted using various multiplicity-of-infection (MOI) of MVA-TRICOM and the efficiency of the vector to enhance the expression of the encoded transgenes was evaluated by FACS analysis. The infection of B-CLL cells with MVA-TRICOM markedly increased the expression of B7-1, ICAM-1 and LFA-3 on the cell surface. The ability of the MVA-TRICOM-infected B-CLL cells to activate specific T cells was subsequently analyzed in proliferation assays, cytotoxic, and cytokine production assays. Proliferation of both autologous and allogeneic T cells was observed when MVA-TRICOM-infected B-CLL cells were used as stimulators in mixed lymphocyte reactions. Autologous CTLs were then generated in vitro using MVA-TRICOM-infected B-CLL cells as APC; these CTLs were capable of lysing uninfected autologous as well as allogeneic B-CLL cells. From our results we conclude that MVA-TRICOM-infected B-CLL cells can be used to induce B-CLL-specific CTLs that, in turn, can mediate the lysis of unmodified B-CLL cells. Our findings have implications for a potential use of MVA-TRICOM-infected B-CLL cells as tumor vaccines in the immunotherapy of B-CLL.

Molecular Mechanism of Nef-Mediated Inhibition of T Cell Migration in Response to SDF-1α.

Lymphocyte trafficking comprises an intricate multistep process, in which lymphocyte surface integrins, endothelial adhesion molecules, and chemokines produced in the local microenvironment play key roles. We previously reported that HIV-1 nef inhibits T cell chemotaxis in response to the physiological ligand SDF-1αwithout altering CXCR4 expression. Here, we explore the mechanism of this effect. HIV-1 nef significantly reduced the expression of LFA-1, inhibited the adhesion of nef-expressing Jurkat T cells to the HUVEC endothelium, and reduced their transendothelial migration. To gain further insight into how nef mediated these effects, substitution or deletion mutations were introduced into the nef gene, and transwell and transendothelial migration assays were performed. Deletion of the N-terminus proximal basic-rich domain did not change the nef-mediated inhibitory effects on T cell migration, whereas removal of the myristoylation site or the proline-rich amino acid sequence motif of Nef abolished these effects. Similarly, HIV-1 nef blocked the transendothelial migration of Jurkat T cells across the HUVEC monolayer. However, the observed inhibition of transendothelial migration conferred by the wild type nef expression was significantly but not completely reduced by introduction of mutations in the myristoylation site and proline-rich motif, indicating the involvement of other regions in the nef gene. Taken together, these results demonstrate that HIV-1 nef can impair the transendothelial migration of T cells in response to SDF-1αby disturbing different steps in the process, and that the myristoylation site and/or proline-rich motif are key to this effect. These data help to establish the molecular mechanisms of nef-mediated impairment of the T cell migratory response, an essential component of host defense, and provide the basis for targeted therapeutic interventions in patients with AIDS.

Differences in the Expression of Adhesion Molecules and Mobilization Efficacy in Healthy Stem Cell Donors and Patients with Hematologic Malignancies.

Stem cell mobilization is achieved by short term administration of G-CSF in healthy donors and G-CSF +/− chemotherapy in patients with malignant diseases. It is known that the crosstalk between adhesion molecules, bone marrow microenvironment, and cytokines facilitates the multi step process of stem cell mobilization from bone marrow to peripheral blood. But still, the biological basis of the large variability in mobilization efficacy remains unclear. The aim of the present study was to evaluate if poor and good mobilizers - healthy donors as well as patients with hematologic malignancies - show differences in the expression of adhesion molecules (VLA-4, L-selectin, LFA-1, PECAM-1, CD44), the chemokine receptor CXCR4 and the G-CSF receptor measured by flow cytometry on CD34 positive cells. Therefore, we investigated 200 aphereses from 132 healthy PBSC donors and 68 patients with a 4-color staining: CD34/CD45 combined with two different adhesion molecules in each sample. The quantitative (mean fluorescence intensity) and qualitative (%) antigen expression was assessed. Donors/patients were divided into three groups according to the peripheral CD34-positive cell count at the day of apheresis: ≤ 30/μl (poor mobilizer; 36 donors, 30 pts.), 31–100/μl (standard mobilizer; 56 donors, 23 pts.), > 100/μl (good mobilizer; 40 donors, 15 pts.). The quantitative antigen expression was significantly higher for LFA-1 in poor vs. good mobilizing donors (GeoMean: 22 vs. 17; p=0.007) and patients (26 vs. 18; p<0.033) and for PECAM-1 (419 vs. 240; p<0.019; only in patients). In contrast, L-selectin showed a significantly lower expression in poor vs. good mobilizing donors (55 vs. 101; p=0.006). Considering the percentage of antigen positivity, LFA-1 was expressed in a significantly higher proportion of CD34 positive cells in poor mobilizing donors (66% vs. 53%; p=0.002) as well as patients (82% vs. 51%; p<0.011). In contrast, CXCR4 and L-selectin expression was significantly lower in samples of poor mobilizing patients (48 % vs. 62%, p<0.001; 90% vs. 95%, p=0.025). VLA-4, PECAM-1, and CD44 were expressed in 100% of CD34 positive cells independent of mobilization capacity. Comparing healthy donors and patients, the qualitative and quantitative adhesion molecule expression is much lower in donors vs. patients for almost all tested antigens and independent of mobilization efficiency. In contrast, the chemokine receptor CXCR4 has a significantly higher expression in CD34+ cells of healthy donors on the one hand, suggesting its downregulation during recovery from myeloablative chemotherapy in patients on the other hand. In summary, our analyses suggest that a higher LFA-1 expression is correlated with a reduced mobilization efficacy in healthy donors as well as patients with hematologic malignancies. The expression of the other tested antigen seems to be regulated differently in donors and patients.

E-Selectin Early Overexpression Induced by Allogeneic Activation in Isolated Mouse Lung

The interaction between host lymphocytes and graft endothelial cells plays an important role in graft rejection. Using our model of isolated ventilated lung from female mouse perfused with fresh blood from either isogeneic or allogeneic male mouse for 3 hours without noticeable ischemia, we have investigated the kinetics of the early events after endothelial cell triggering by E-selectin engagement. Isogeneic perfusion induced nonspecific endothelial cell activation, which was characterized by up-regulation of E-selectin, intercellular adhesion molecule (ICAM)-1, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, and lymphotoxin-alpha (mRNAs by real-time polymerase chain reaction). Allogeneic perfusion was characterized after 3 hours by an additional loose adhesion of lymphocytes mediated by the E-selectin and related to the allogeneic activation of endothelial cells. These in turn expressed the I-A molecule (immunostaining). ICAM-1 and lymphocyte function-associated antigen (LFA)-3 mRNA levels were significantly increased in lung extracts after 2 hours, then vascular cell adhesion molecule (VCAM)-1 and TNF-alpha mRNAs after 3 hours without evidence of TNF-alpha production (enzyme-linked immunoadsorbent assay). The major participation of the E-selectin in early allogeneic activation by way of the protein kinase (PK)C pathway was confirmed by using a neutralizing anti-CD62E monoclonal antibody or the inhibitory PKC 19-31 fragment. Altogether, our results demonstrate that E-selectin expression (1) is not a consequence of TNF-alpha triggering, (2) up-regulates its own expression and expression of I-A, VCAM-1, TNF-alpha, and lymphotoxin-alpha mRNAs, and (3) down-regulates expression of LFA-3 and ICAM-1 mRNAs. In conclusion, in our physiologic model, the E-selectin highly participates in the loose adhesion of allogeneic lymphocytes and in the early activation of endothelial cell and therefore in structural and functional lung alterations.

Incubation of bovine PMNs with conditioned medium from BHV-1 infected peripheral blood mononuclear cells increases their susceptibility to Mannheimia haemolytica leukotoxin

Active infection with bovine herpesvirus-1 (BHV-1) increases the susceptibility of cattle to secondary bacterial pneumonia with Mannheimia (Pasteurella) haemolytica A1. In the present study we found that bovine PMNs incubated with conditioned media from BHV-1 infected peripheral blood mononuclear cells (PBMCs) exhibited increased LFA-1 expression, enhanced LKT binding and increased LKT cytotoxicity. These effects were abrogated when the conditioned medium was pre-incubated with an anti-IL-1beta Mab before being added to the PMNs. These findings suggest that BHV-1 infection, and the resulting release of IL-1beta and perhaps other inflammatory cytokines, can stimulate activation of LFA-1 in bystander bovine PMNs, thus enhancing the binding and biological effects of LKT.

Lymphocyte subsets and adhesion molecule expression in milk and blood of periparturient dairy cattle.

Fifteen Holstein dairy cattle were monitored for lymphocyte subsets and expression of adhesion molecules on cells in milk and blood at parturition and at intervals up to 21 days post-partum. Using flow cytometry, we determined percentages of T cells (CD4+, CD8+, gammadelta) and B cells from milk and blood of these cows. We also measured expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on lymphocytes in milk and blood. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points while significantly higher percentages of B cells were found in blood than in milk at all time points. There were minimal to no significant differences in percentages of CD4+ or gammadelta+ cells between milk and blood. Expression of adhesion molecules was consistently higher on all subsets of milk lymphocytes compared with blood lymphocytes. These differences were most pronounced and statistically significant at calving and in the first week following calving. CD62L, LPAM-1 and CD44 were expressed on a significantly higher percentage of lymphocytes in milk at calving than in milk at subsequent sampling times, while LFA-1 expression on lymphocytes in milk was significantly lower at calving than at subsequent times.

Effect of ligustrazine on the expression of LFA-1, ICAM-1 following bone marrow transplantation in mice.

The effects of ligustrazine on the expression of LFA-1, ICAM-1 in bone marrow tissue and the mechanism promoting hematopoietic reconstitution following bone marrow transplantation (BMT) were investigated. The 150 mice were randomly divided into 3 groups: normal group, saline group and ligustrazine group. The normal group received no treatment, while in the saline group and ligustrazine group, the mice were subjected to normal saline (0.2 ml, twice a day) and ligustrazine (0.2 ml, twice a day) respectively through a gastric tube. At the 7th, 14th, 21st and 28th day after BMT, survival rate, colony forming unit of spleen (CFU-S), peripheral blood cells and bone marrow mononuclear cells (BMMNC) were measured, histological changes in bone marrow tissue were observed and the expression level of LFA-1, ICAM-1 was detected. In ligustrazine group CFU-S counts on the 10th day and the peripheral blood WBC, PLT, BMMNC counts, hematopoietic tissue volume as well as the expression level of LFA-1 on the 7th, 14th, 21st, 28th day after BMT were higher than in saline group (P<0.01 or P<0.05). Mature RBC volume and the expression level of ICAM-1 were significantly lower in the ligustrazine group than in the saline group (P<0.01 or P<0.05). In the ligustrazine group, fat tissue volume was higher on the 7th, 14th day after BMT (P<0.01) and was lower on the 21st, 28th day (P<0.01) after BMT than in the saline group. It was concluded that Ligustrazine could improve bone marrow microenvironment and promote hematopoietic reconstitution.