The cellular members of the interleukin-10 (IL-10) cytokine family share sequence homology with IL-10, whereas their sites of expression and their functions are divergent. One of these factors, AK155 or IL-26, was discovered because of its overexpression in human T lymphocytes after growth transformation by the simian rhadinovirus herpesvirus saimiri. In addition, the gene is transcribed in various types of primary and immortalized T-cells. Here we describe epithelial cells, namely colon carcinoma cells and keratinocytes, as targets of this T-cellular lymphokine. Purified recombinant IL-26 induced the rapid phosphorylation of the signal transducer and activator of transcription factors 1 and 3. As a result, secretion of IL-10 and IL-8, as well as cell surface expression of CD54 were enhanced. Moreover, we show that the IL-26 protein binds to heparin, is released from the cell surface, and can be functionally inhibited by heparin. The sensitivity to recombinant IL-26 of various cell lines strictly correlated with the expression of the long chain of the IL-20 receptor. Because blocking antibodies against either the short chain of the IL-10 receptor or the long chain of the IL-20 receptor inhibited IL-26-dependent signal transduction, and transient expression of these receptor chains induced IL-26 responsivity in non-sensitive cells, we propose that the IL-20 receptor 1 and IL-10 receptor 2 chains participate in forming the IL-26 receptor. Targeting epithelial cells, the T-cell lymphokine IL-26 is likely to play a role in local mechanisms of mucosal and cutaneous immunity.

Inflammatory mediators in the tumour microenvironment promote tumour growth, vascular development and enable evasion of anti-tumour immune responses, by disabling infiltrating dendritic cells. However, the constituents of the tumour microenvironment that directly influence dendritic cell maturation and function are not well characterised. Our aim was to identify tumour-associated inflammatory mediators which influence the function of dendritic cells. Tumour conditioned media obtained from cultured colorectal tumour explant tissue contained high levels of the chemokines CCL2, CXCL1, CXCL5 in addition to VEGF. Pre-treatment of monocyte derived dendritic cells with this tumour conditioned media inhibited the up-regulation of CD86, CD83, CD54 and HLA-DR in response to LPS, enhancing IL-10 while reducing IL-12p70 secretion. We examined if specific individual components of the tumour conditioned media (CCL2, CXCL1, CXCL5) could modulate dendritic cell maturation or cytokine secretion in response to LPS. VEGF was also assessed as it has a suppressive effect on dendritic cell maturation. Pre-treatment of immature dendritic cells with VEGF inhibited LPS induced upregulation of CD80 and CD54, while CXCL1 inhibited HLA-DR. Interestingly, treatment of dendritic cells with CCL2, CXCL1, CXCL5 or VEGF significantly suppressed their ability to secrete IL-12p70 in response to LPS. In addition, dendritic cells treated with a combination of CXCL1 and VEGF secreted less IL-12p70 in response to LPS compared to pre-treatment with either cytokine alone. In conclusion, tumour conditioned media strongly influences dendritic cell maturation and function.

Syndecan-1 (CD138) is a heparan sulfate-bearing proteoglycan present on the surface of myeloma cells where it mediates myeloma cell-cell and cell-extracellular matrix adhesion. In this study, we examined myeloma cell lines for cell membrane localization of syndecan-1. On some cells we note a striking localization of syndecan-1 to a single small membrane protrusion, with the remainder of the cell surface being mostly negative for syndecan-1. Examination of cell morphology reveals that a proportion of cells from myeloma cell lines, as well as primary myeloma cells, are polarized, with a uropod on one end and lamellipodia on the other end. On these polarized cells, syndecan-1 is specifically targeted to the uropod, but in contrast, on nonpolarized cells syndecan-1 is evenly distributed over the entire cell surface. In addition to syndecan-1, several other cell surface molecules localize specifically to the uropod, including CD44 and CD54. Functional assays reveal that myeloma cell lines with a high proportion of polarized cells have a much higher migratory potential than cell lines with few polarized cells. Moreover, the uropod is the cell pole preferentially involved in aggregation of myeloma cells and in adhesion of myeloma cells to osteoblast-like cells. When polarized myeloma cells are incubated with heparin-binding proteins, like hepatocyte growth factor or osteoprotegerin, they concentrate in the uropod. These data indicate that syndecan-1 is targeted to the uropod of polarized myeloma cells and that this targeting plays a role in promoting cell-cell adhesion and may also regulate the biological activity of heparin-binding cytokines.

OBJECTIVE

Fcagamma and complement receptors play an important role in the interaction between immune complexes (IC) and monocytes/macrophages. Recent work has demonstrated that their relative expression on these cells may be modified by cytokines, including TNF-alpha and IL-4. Furthermore, cytokines may alter the expression of adhesion molecules such as ICAM-1. However, little data exist on the in vivo expression of specific Fcgamma and complement receptors in systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), two diseases in which IC are important in pathogenesis.

METHODS

Venous blood was obtained from 30 patients with SLE, 25 with RA and 25 healthy controls. Monocyte phenotype was determined by flow cytometric analysis of whole blood samples, with selective gating using forward and side scatter signals. Surface expression of Fcgamma receptors RI (CD64), RII (CD32) and RIII (CD16), complement receptors CR1 (CD35) and CR3 (CD11b/CD18), and adhesion molecules ICAM-1 (CD54) and CD11a (LFA-1) was determined. The effects of disease activity and corticosteroid therapy on the expression of these molecules were also examined.

RESULTS

The expression of FcgammaRII was reduced on monocytes from patients with SLE compared with healthy controls and patients with RA (P = 0.002). This did not correlate with disease activity using conventional indices [SLEDAI (SLE disease activity index), C3/C4 levels and anti-double-stranded DNA antibody titres], and was independent of prednisolone therapy. There was no significant difference in FcgammaRI or RIII expression on SLE monocytes compared with healthy controls. In contrast, the expression of FcgammaRIII was increased on RA monocytes (P = 0.01), this being highest in patients with active disease. The proportion of FcgammaRIII-positive monocytes was also increased in RA, and prednisolone therapy was associated with a lower proportion of FcgammaRIII-positive cells. An increase in CR3 expression was seen on RA monocytes (P = 0.002), whilst CR1 was increased on monocytes from patients with active SLE or active RA. ICAM-1 expression was reduced on monocytes from patients with SLE (P = 0.002), although high-dose prednisolone therapy was associated with the lowest level of surface ICAM-1 on monocytes.

CONCLUSIONS

Peripheral blood monocytes from patients with SLE or RA display significantly altered phenotypes compared with those from healthy controls. The observed reduction in SLE of FcgammaRII may represent a mechanism by which monocytes are protected from IC-mediated activation. Prednisolone therapy and disease activity had little effect on phagocytic receptor expression. The observed changes may reflect the different cytokine profiles seen in SLE and RA.

CD40 is a membrane differentiation antigen constitutively expressed on B cells that induces B cell growth and Ig synthesis after ligation with anti-CD40 mAb or with the recently identified CD40 ligand (CD40L). CD40L is rapidly induced on T cells after activation with anti-CD3 mAb or mitogens. While CD40-CD40L interactions are clearly beneficial to B cells, we speculated that a reciprocal costimulation of T cells might also occur. We have used genetic transfection to demonstrate that interactions between human small, resting T cells and CD40+ murine transfectants substantially augmented anti-CD3 induced T cell proliferation and resulted in the generation of CTL. T cell proliferation costimulated by CD40 was IL-2 dependent. The ability of CD40+ transfectants to costimulate T cell proliferation was specific in that VCAM-1+, CD54+, CD72+, CD56+, CD31+, and fas+ transfectants in the same host cells were inactive. CD4+ T cells preferentially responded to CD40 costimulation, whereas CD8+ T cells were substantially less reactive. By contrast, costimulation with B7 transfectants induced equivalent proliferation in the CD4+ and CD8+ T cell subsets. In addition, adult naive and memory T cells, as well as cord blood T cells, were responsive to CD40. These findings suggest that the CD40-CD40L costimulation pathway may allow for selective expansion of CD4+ T cells after interaction with CD40-bearing APC. The relatively restricted expression of CD40 on APC, as well as on medullary and cortical thymic epithelium, indicates a possible role for this interaction in T cell differentiation and activation.

Receptor-interacting protein (RIP) has been reported to associate with tumor necrosis-associated factor (TRAF)2 and TRAF6. Since TRAF2 and TRAF6 play important roles in CD40 signaling and TRAF6 plays an important role in TLR4 signaling, we examined the role of RIP in signaling via CD40 and TLR4. Splenocytes from RIP(-/-) mice proliferated and underwent isotype switching normally in response to anti-CD40-IL-4 but completely failed to do so in response to LPS-IL-4. However, they normally up-regulated TNF-alpha and IL-6 gene expression and CD54 and CD86 surface expression after LPS stimulation. RIP(-/-) splenocytes exhibited increased apoptosis and impaired Akt phosphorylation after LPS stimulation. These results suggest that RIP is essential for cell survival after TLR4 signaling and links TLR4 to the phosphatidylinositol 3 kinase-Akt pathway.

Signals generated through CD28-B7 and CD40 ligand (CD40L)-CD40 interactions have been shown to be crucial for the induction of long-term allograft survivability. We have recently demonstrated that humanized anti-CD40L (hu5C8) prevents rejection of mismatched renal allografts in primates. To investigate potential mechanisms of CD40L-induced allograft acceptance, we coimmobilized hu5C8 with suboptimal amounts of anti-CD3 to stimulate CD4(+) T cells. We now report that anti-CD3/CD40L costimulation results in CD28-independent activation and subsequent deletion of resting T cells. Coligation of CD3 and CD40L increased expression of CD69, CD25, and CD54 on CD4(+) T cells. We also found that costimulation with anti-CD3/CD40L resulted in enhanced production of interleukin (IL)-10, interferon gamma, and tumor necrosis factor alpha but not IL-2 or IL-6. Interestingly, after several days, anti-CD3/CD40L-mediated activation was followed by apoptosis in a significant population of cells. Consistent with that observation, anti-CD3/CD40L did not enhance the antiapoptotic proteins Bcl-2 and Bcl-xL. Further, the addition of CD28 at 24 h failed to rescue those cells induced to die after costimulation with anti-CD3/CD40L. Together, these data suggest that the graft-sparing effect of hu5C8 in vivo may result in part from early and direct effects on CD4(+) T cells, including a vigorous induction of immunomodulatory cytokines and/or apoptosis of allograft-specific T cells.

In a group of 30 human tumors, comprising 12 lung, 14 ovarian, 2 breast carcinomas, 1 hypernephroma and 1 mid-gut carcinoid, the expression of major histocompatibility complex (MHC) class I molecules and the intercellular adhesion molecule 1 (CAM-1, CD54) was found to vary independently. Some tumors expressed both or neither of these molecules. Among 9/13 ICAM-1+ tumors, in which greater than 50% cells reacted with the anti-ICAM-1 monoclonal antibody (mAb) (LB-2), the class I antigen was also detected on greater than 50% of the cells. Only 2 ICAM-1+ tumors were class-I-. In 5/17 cases the tumors were MHC-class-I+ and ICAM-1-. Lymphocytes collected from the blood or from the tumor site were assayed for recognition on the tumor cells in the auto-tumor cytotoxicity test and in mixed lymphocyte tumor cell culture (MLTC). Positive results were obtained only with the MHC-class-I+/ICAM-1+ tumors. In vitro treatment of the tumor cell suspensions with interferon gamma and tumor necrosis factor alpha (TNF alpha) induced or enhanced the ICAM-1 and/or class I antigen expression in 8/12 cases. Of the tumor samples treatged, 8/9 acquired stimulatory capacity and 3/10 became susceptible to lysis by the lymphocytes. In 6/6 MLTC performed with the cytokine-treated tumor cells, cytotoxicity against the autologous tumor was generated. Three of these MLTC lymphocytes also lysed the untreated targets. mAb directed to class I antigens or to ICAM-1 inhibited both the stimulation by and the lysis of tumor cells when confronted with fresh lymphocytes. The cytotoxicity generated in the MLTC was also inhibited. If, however, the cytotoxic function was induced in MTLC containing interleukin-2 (5 U/ml), inhibition was obtained only by pretreatment of the targets with mAb against ICAM-1. The results show thus (a) that the lymphocytes react in vitro with tumor cells only if these express both MHC class I molecules and ICAM-1; (b) that expression of these molecules can be induced by interferon alpha and TNF alpha; (c) that cytotoxic effectors generated in the MLTC with cytokine-treated tumors can also act on the untreated tumor cells. The requirement of the two surface moieties for the interaction with lymphocytes was also substantiated by blockade with relevant mAb.

Toll-like receptor (TLR) agonists are considered adjuvants in clinical trials of cancer immunotherapy. Here, we investigated the modulation of gammadelta T cell-mediated tumor cell lysis by TLR ligands. gammadelta T-cell cytotoxicity and granzyme A/B production were enhanced after pretreatment of tumor cells with TLR3 [poly(I:C)] or TLR7 ligand (imiquimod). We examined TLR3- and TLR7-expressing pancreatic adenocarcinomas, squamous cell carcinomas of head and neck and lung carcinomas. Poly(I:C) treatment of pancreatic adenocarcinomas followed by coculture with gammadelta T cells resulted in an upregulation of CD54 on the tumor cells. The interaction of CD54 and the corresponding ligand CD11a/CD18 expressed on gammadelta T cells is responsible for triggering effector function in gammadelta T cells. Moreover, treatment with imiquimod downregulated MHC class I molecules on tumor cells possibly resulting in a reduced binding affinity for inhibitory receptor NKG2A expressed on gammadelta T cells. These results indicate that TLR3 or TLR7 ligand stimulation of tumor cells enhances the cytotoxic activity of expanded gammadelta T cells of cancer patients in vitro.

Ewing's family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlled EWS/ETS-inducible system in human bone marrow-derived mesenchymal progenitor cells (MPCs). Ectopic expression of both EWS/FLI1 and EWS/ERG proteins resulted in a dramatic change of morphology, i.e., from a mesenchymal spindle shape to a small round-to-polygonal cell, one of the characteristics of EFT. EWS/ETS also induced immunophenotypic changes in MPCs, including the disappearance of the mesenchyme-positive markers CD10 and CD13 and the up-regulation of the EFT-positive markers CD54, CD99, CD117, and CD271. Furthermore, a prominent shift from the gene expression profile of MPCs to that of EFT was observed in the presence of EWS/ETS. Together with the observation that EWS/ETS enhances the ability of cells to invade Matrigel, these results suggest that EWS/ETS proteins contribute to alterations of cellular features and confer an EFT-like phenotype to human MPCs.

Dietary gluten has been associated with an increased risk of type 1 diabetes. We have evaluated inflammation and the mucosal immune response to gliadin in the jejunum of patients with type 1 diabetes. Small intestinal biopsies from 17 children with type 1 diabetes without serological markers of celiac disease and from 50 age-matched control subjects were examined by immunohistochemistry. In addition, biopsies from 12 type 1 diabetic patients and 8 control subjects were cultured with gliadin or ovalbumin peptic-tryptic digest and examined for epithelial infiltration and lamina propria T-cell activation. The density of intraepithelial CD3(+) and gammadelta(+) cells and of lamina propria CD25(+) mononuclear cells was higher in jejunal biopsies from type 1 diabetic patients versus control subjects. In the patients' biopsies cultured with peptic-tryptic gliadin, there was epithelial infiltration by CD3(+) cells, a significant increase in lamina propria CD25(+) and CD80(+) cells and enhanced expression of lamina propria CD54 and crypt HLA-DR. No such phenomena were observed in control subjects, even those with celiac disease-associated HLA haplotypes. In conclusion, signs of mucosal inflammation were present in jejunal biopsies from type 1 diabetic patients, and organ culture studies indicate a deranged mucosal immune response to gliadin.

Autotaxin (ATX/NPP2) is a tumor cell motility-stimulating factor that displays both a nucleotide pyrophosphatase/phosphodiesterase activity and a recently described lysophospholipase D (lysoPLD) activity. The precise function of ATX in tumor cells and the role of ATX in thyroid carcinoma remains unclear. We have quantified ATX mRNA expression in thyroid carcinoma cell lines and in tissues of patients with thyroid carcinomas. ATX gene activity was significantly higher in undifferentiated anaplastic thyroid carcinoma cell lines (UTC) and tumor tissues as compared to follicular thyroid carcinoma (FTC) cell lines, FTC tissues or goiter tissues that were used as a control. In the thyroid carcinoma cell line 1736, EGF and bFGF stimulated ATX mRNA expression, whereas the cytokines IL-4, IL-1beta and TGF-beta reduced ATX transcriptional levels. FTC-133 cells, stably transfected with an expression vector for ATX, showed a higher lysoPLD activity, a higher proliferation rate and an increased migratory behavior. In addition, ATX also displayed a paracrine stimulatory effect on the motility of different thyroid carcinoma cell lines. Overexpression of ATX in the stably transfected FTC-133 resulted in down-regulation of CD54/ intercellular adhesion molecule-1 (ICAM-1) gene expression and augmented gene activity of the pro-angiogenic chemokine IL-8. We conclude that ATX may be regarded as a new tissue marker for undifferentiated human thyroid carcinoma cells. ATX increases the proliferation and migration of thyroid carcinoma cell lines and may also affect the angiogenic potential of thyroid carcinoma cells. Further studies are needed to provide insight into the role of ATX in the normal and neoplastic thyroid gland.

IL-27 is a novel heterodimeric cytokine of the IL-12 family that plays an important role in the regulation of T cell responses. Its role on human B cells has not been previously studied. In this study, we show that both chains of the IL-27 receptor complex, IL-27R and gp130, are constitutively expressed at the surface of naive and memory human tonsillar B cells, and are induced on germinal center B cells following CD40 stimulation. In naive B cells, IL-27 induced strong STAT1 and STAT3 phosphorylation, whereas it induced moderate STAT1 and low STAT3 activation in memory B cells. IL-27 induced T-bet expression in naive and memory B cells stimulated by CD40 or surface Ig engagement, but induced significant IL-12Rbeta2 surface expression in anti-Ig-stimulated naive B cells only. In anti-Ig-stimulated naive or memory B cells, IL-27 also induced CD54, CD86, and CD95 surface expression. In addition, IL-27 increased proliferation of anti-Ig-activated naive B cells and of anti-CD40-activated naive and germinal center B cells, but not of CD40-activated memory B cells. These data indicate that the B cell response to IL-27 is modulated during B cell differentiation and varies depending on the mode of B cell activation.

Microglial cells can be derived directly from the dissociated brain tissue by sorting procedures, from postnatal glial cultures by mechanic isolation or from pluripotent stem cells by differentiation. The detailed molecular phenotype of microglia from different sources is still unclear. Here, we performed a whole transcriptome analysis of flow cytometry-sorted microglia, primary postnatal cultured microglia, embryonic stem cell derived microglia (ESdM), and other cell types. Microglia and ESdM, both cultured in serum-free medium, were closely related to sorted microglia and showed a unique transcriptome profile, clearly distinct to other myeloid cell types, T cells, astrocytes, and neurons. ESdM and primary cultured microglia showed strong overlap in their transcriptome. Only 143 genes were differentially expressed between both cell types, mainly derived from immune-related genes with a higher activation status of proinflammatory and immune defense genes in primary microglia compared to ESdM. Flow cytometry analysis of cell surface markers CD54, CD74, and CD274 selected from the microarray confirmed the close phenotypic relation between ESdM and primary cultured microglia. Thus, assessment of genome-wide transcriptional regulation demonstrates that microglial cells are unique and clearly distinct from other macrophage cell types.

Intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and for function of B lymphocytes as antigen-presenting cells. ICAM-1 expression is upregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving to render antigen-stimulated B cells more receptive to T cell-mediated costimulatory signals. We have investigated BCR-induced expression of the Icam-1 gene in primary B cells and B cell lines and have found it to be dependent on BCR-induced expression of the transcription factor EGR1. Icam-1 transcription, induced by BCR cross-linking or bypassing the BCR with phorbol ester, is absent in a B cell line in which the EGR1-encoding gene (egr-1) is methylated and not expressed. A potential EGR1-binding site was located at -701 bp upstream of the murine Icam-1 gene transcription start site and shown by electrophoretic mobility shift assay to bind to murine EGR1. Mutation of this site in the context of 1.1 kb of the Icam-1 promoter significantly abrogated transcriptional induction by phorbol ester and anti-mu stimulation in primary B cells. A direct effect of EGR1 on the Icam-1 promoter is suggested by the ability of EGR1 expressed from an SV40-driven expression vector transactivate the wild-type Icam-1 promoter, whereas mutation of the EGR1 mutation of the EGR1 binding motif at -701 bp markedly compromises this induction. These data identify EGR1 as a signaling intermediate in BCR-stimulated B cell functional responses, specifically linking BCR signal transduction to induction of the Icam-1 gene. Furthermore, similar findings for BCR-induced CD44 gene induction (Maltzman, J.S., J.A. Carman, and J.G. Monroe. 1996. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. Cell. Biol. In press) suggest that EGR1 may be an important signaling molecule for regulating levels of migration and adhesion molecules during humoral immune responses.

We have isolated low-density, nonadherent, nonphagocytic, HLA-DR+ve cells with the morphology of dendritic cells (DCs) from the cord blood of full-term newborn infants. Relative to adult DCs, cord blood DCs were poor stimulators of the mixed leukocyte reaction when either adult or cord blood mononuclear cells (MNCs) or T lymphocytes were used as responder cells. In contrast, cord blood T cells and MNCs responded normally to allogeneic adult DCs. Cord blood DCs performed poorly as accessory cells for T-lymphocyte mitogenic responses at suboptimal concentrations of concanavalin A (Con A) and phytohemagglutinin A or at optimal concentrations of mitogen and low numbers of DCs. Addition of recombinant interleukin-2 (rIL-2) or recombinant interferon-gamma (rIFN-gamma) to cord blood DC-T-cell cultures containing a suboptimal concentration of Con A potentiated the proliferative response. In contrast, rIL-2 and rIFN-gamma exerted little effect on the proliferative response of adult T cells cultured with Con A and DCs. Flow cytometric studies showed that levels of intercellular adhesion molecule-1 (ICAM-1; CD54) and major histocompatibility complex (MHC) class I HLA-ABC and class II HLA-DR antigens on cord blood DCs were significantly lower than those on adult blood DCs. These findings suggest that the relative inefficiency of cord blood DCs in the activation of T cells may be related to their low cell surface expression of MHC and cell adhesion molecules. The demonstrated impairment of cord blood DC function could be of importance in understanding the immunologic relationship between the fetus and mother and could contribute to the susceptibility of newborns to infection.

Ixodes scapularis ticks transmit Anaplasma phagocytophilum (Ap), agent of human granulocytic anaplasmosis (HGA). Invasion of neutrophil granulocytes (PMN) by Ap is the hallmark of the disease, but these short-lived phagocytes are not likely the sole cell type required for productive infection. We analyzed infection of microvascular endothelial cells during pathogenesis of anaplasmosis in vivo and in vitro. Organs from Ap-infected mice were processed for confocal microscopy 41 days p.i. Fluorescent labeling of heart and liver sections using anti-factor VIII and anti-MSP2 antibodies allowed colocalization of Ap and vascular endothelium, indicating infection. Ap rapidly invaded and grew within HMEC-1 human microvascular endothelial cells and readily transferred to PMN. Over 50% of PMN became infected within two hours of coincubation with HMEC-1. PMN adhered to, polarized, and migrated upon infected endothelial monolayers. The Ap receptor on human PMN is PSGL-1, and infected endothelial cells upregulate ICAM-1 (CD54), but the mechanisms of transfer of Ap remain unknown. To elucidate the cellular determinants involved, we tested relevant antibodies and lectins. Anti-PSGL-1 reduced infection of PMN, but did not inhibit adherence of PMN to Ap infected HMEC-1 cells while anti-CD18 did. Sialidase pretreatment increased, and EDTA and fucoidan decreased binding of Ap to HMEC-1, whereas several other lectins had no effect. An endothelial reservoir of Ap offers opportunities for ongoing, direct cell-to-cell infection of PMN, avoidance of host immune effectors, and completion of the Ap life cycle by infection of circulating leukocytes available for transfer to blood-feeding ticks.

T cells and other leucocytes regularly occur within and subjacent to the gut epithelium. Recent data suggest that intestinal epithelial cells may exert accessory immunological functions. Although interactions between leucocytes and accessory cells usually require expression of adhesion molecules, intestinal epithelium has generally been considered negative for intercellular adhesion molecule-1 (ICAM-1) by immunohistochemical techniques. We therefore studied the expression of ICAM-1 and lymphocyte function-associated antigen-3 (LFA-3) by two colonic epithelial cell lines and found that both adhesion molecules were constitutively present at low levels. ICAM-1 protein expression could be enhanced within 4 h by cytokines, particularly after co-incubation with interferon-gamma (IFN) and tumour necrosis factor-alpha (TNF), interleukin-1 beta (IL-1), or IL-6. IFN also resulted in accumulation of ICAM-1 mRNA. Conversely, the LFA-3 expression was virtually unaffected by cytokine stimulation. These data imply that intestinal epithelial cells under certain conditions may bear adhesion molecules required for cooperation with juxtaposed leucocytes in situ, and that the expression of some of these molecules is modulated by cytokines from activated mucosal leucocytes.

Lassa virus (LV) and Mopeia virus (MV) are closely related members of the Arenavirus genus, sharing 75% amino acid sequence identity. However, LV causes hemorrhagic fever in humans and nonhuman primates, whereas MV cannot induce disease. We have previously shown that antigen-presenting cells (APC)-macrophages (MP) and dendritic cells (DC)-sustain high replication rates of LV but are not activated, suggesting that they play a role in the immunosuppression observed in severe cases of Lassa fever. Here, we infected human APC with MV and analyzed the cellular responses induced. MV infection was productive in MP and even more so in DC. Apoptosis was not induced in either cell type. Moreover, unlike DC, MP were early and strongly activated in response to MV, as shown by the increased surface expression of CD86, CD80, CD54, CD40, and HLA-abc and by the production of mRNA encoding alpha interferon (IFN-alpha), IFN-beta, tumor necrosis factor alpha and interleukin-6. In addition, MV-infected MP produced less of the virus than DC, which was related to the fact that these cells secreted IFN-alpha. Thus, the strong activation of MP is probably a major event in the control of MV infection and may be involved in the induction of an adaptive immune response in infected hosts. These results may explain the difference in pathogenicity between LV and MV.

Cysteine proteinases (gingipains) elaborated from Porphyromonas gingivalis exhibit enzymatic activities against a broad range of host proteins and are considered key virulence factors in the onset and development of adult periodontitis and host defense evasion. In this study, we examined the ability of arginine-specific gingipains (high molecular mass Arg-specific gingipain (HRGP) and Arg-specific gingipain 2) and lysine-specific gingipain (KGP) to cleave monocyte CD14, the main receptor for bacterial cell surface components such as LPS. Binding of anti-CD14 mAb MY4 to human monocytes was almost completely abolished by 0.3 microM HRGP and KGP treatments for 15 min, and 1 microM RGP2 for 30 min. In contrast, the expressions of Toll-like receptor 4, and CD18, CD54, CD59, and HLA-A, -B, -C on monocytes were slightly increased and decreased, respectively, by 0. 3 microM HRGP and KGP. This down-regulation resulted from direct proteolysis, because 1) gingipains eliminated MY4 binding even to fixed monocytes, and 2) CD14 fragments were detected in the extracellular medium by immunoblot analysis. Human rCD14 was degraded by all three gingipains, which confirmed that CD14 was a substrate for gingipains. TNF-alpha production by monocytes after HRGP and KGP treatments was decreased at 1 ng/ml, but not at 20 microg/ml LPS, indicating that gingipains inhibited a CD14-dependent cell activation. These results suggest that gingipains preferentially cleave monocyte CD14, resulting in attenuation of the cellular recognition of bacteria, and as a consequence sustain chronic inflammation.

It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

We examined 26 patients with human immunodeficiency virus-1 (HIV-1)-associated Kaposi's sarcoma (KS), and 76 HIV-1-infected (HIV-1+) people without KS or uninfected (HIV-1-) controls for the presence of circulating KS-like spindle cells. Adherent cells that had spindle morphology and several characteristics of spindle cells of KS lesions (KS cells) were identified in the peripheral blood mononuclear cell fraction only after culture in the presence of conditioned medium (CM) from activated lymphocytes. The peripheral blood-derived spindle cells (PBsc) expressed a variety of endothelial cell markers, such as Ulex europaeus I lectin, EN4, EN2/3, EN7/44, CD13, CD34, CD36, CD54, ELAM-1, and HLA-DR. However, they were negative for CD2, CD19, PaIE, and factor VIII-related antigen. The PBsc produced angiogenic factors as evidenced by the ability of CM from these cells to promote growth of normal vascular endothelial cells. In addition, subcutaneously injected PBsc stimulated angiogenesis in vivo in athymic nude mice. We determined that the number of PBsc grown from the peripheral blood of HIV-1+ patients with KS or at high risk to develop KS were increased by 78-fold (P = .0001) and 18-fold (P = .005), respectively, when compared with HIV-1- controls. The number of spindle cells cultured from the HIV-1+ patients at low risk for developing KS, eg, HIV-1+ injection drug users, showed no statistical increase when compared with HIV-1- controls. The presence of increased PBsc with characteristics of KS cells in HIV-1+ KS patients or patients at high risk for developing KS gives insights into the origin of KS cells and may explain the multifocal nature of the disease. In addition, this may be useful in predicting the risk of KS development.

Leukostasis and tissue infiltration by leukemic cells are poorly understood life-threatening complications of acute leukemia. This study has tested the hypothesis that adhesion receptors and cytokines secreted by blast cells play central roles in these reactions. Immunophenotypic studies showed that acute myeloid leukemia (AML) cells (n = 78) of the M0 to M5 subtypes of the French-American-British Cooperative Group expressed various amounts of adhesion receptors, including CD11a, b, c/CD18, CD49d, e, f/CD29, CD54, sCD15, and L-selectin. The presence of functional adhesion receptors was evaluated using a nonstatic adhesion assay. The number of blast cells attached to unactivated endothelium increased by 7 to 31 times after a 6-hour exposure of endothelium to tumor necrosis factor (TNF)-alpha. Inhibition studies showed that multiple adhesion receptors--including L-selectin, E-selectin, VCAM-1, and CD11/CD18--were involved in blast cell adhesion to TNF-alpha-activated endothelium. Leukemic cells were then cocultured at 37 degrees C on unactivated endothelial cell monolayers for time periods up to 24 hours. A time-dependent increase in the number of blasts attached to the endothelium and a concomitant induction of ICAM-1, VCAM-1, and E-selectin were observed. Additional experiments revealed that endothelial cell activation by leukemic myeloblasts was caused by cytokine secretion by blast cells, in particular TNF-alpha and IL-1 beta, and direct contacts between adhesion receptors expressed by blast cells and endothelial cells. Thus, leukemic cells have the ability to generate conditions that promote their own adhesion to vascular endothelium, a property that may have important implications for the pathophysiology of leukostasis and tissue infiltration by leukemic blast cells. (Blood. 2001;97:2121-2129)

Human myoblasts, cultured from muscle and purified to greater than 95%, were investigated for their capacity to act as facultative APC. The myoblasts reacted with antidesmin mAb and had the capacity to fuse into multinucleated myotubes in appropriate medium. The expression of HLA class I, HLA-DR, HLA-DP, HLA-DQ, intercellular adhesion molecule-1 (ICAM-1/CD54), lymphocyte function-associated (LFA) molecules LFA-1 (CD11a/CD18), LFA-2 (CD2), and LFA-3 (CD58) was investigated by FACS analysis before and after induction for various times with human rIFN-gamma, TNF-alpha, or both. Without cytokine induction, myoblasts expressed only HLA-class I and LFA-3. IFN-gamma alone or in combination with TNF-alpha induced the expression of HLA-DR and ICAM-1 reaching a plateau after 48 h, followed by HLA-DP and even later HLA-DQ. TNF-alpha alone induced only ICAM-1. The functional capacity of myoblasts to present Ag to CD4+ T cells was investigated using autologous T cell lines specific for tuberculin, tetanus toxoid, and human myelin basic protein. Noninduced myoblasts or myoblasts treated with TNF-alpha alone could not present any of these Ag to the T cells. However, myoblasts treated with IFN-gamma induced Ag-specific proliferation. In the presence of relevant Ag, myoblasts were killed by the T cells as observed by microscopy and measured by 51Cr release. Ag-specific T cell proliferation and myoblast killing was inhibited in the presence of anti-DR mAb. These results suggest that human myoblasts may act as facultative APC during local immune reactions in muscle.