Inflammatory sites, such as rheumatoid arthritis (RA) synovial tissue, contain large numbers of activated B cells and plasma cells. However, the mechanisms maintaining B cell viability and promoting their differentiation are not known, but interactions with stromal cells may play a role. To examine this, purified human peripheral B cells were cultured with a stromal cell line (SCL) derived from RA synovial tissue, and the effects on apoptosis and expression of Bcl-2-related proteins were analyzed. As a control, B cells were also cultured with SCL from osteoarthritis synovium or skin fibroblasts. B cells cultured with medium alone underwent spontaneous apoptosis. However, B cells cultured with RA SCL cells exhibited less apoptosis and greater viability. Although SCL from osteoarthritis synovium and skin fibroblasts also rescued B cells from apoptosis, they were less effective than RA SCL. B cell expression of Bcl-xL was markedly increased by RA SCL in a contact-dependent manner, whereas B cell expression of Bcl-2 was unaffected. Protection of B cells from apoptosis and up-regulation of Bcl-xL by RA SCL were both blocked by mAbs to CD106 (VCAM-1), but not CD54 (ICAM-1). Furthermore, cross-linking of CD49d/CD29 (very late Ag-4) on the surface of B cells rescued them from apoptosis and up-regulated Bcl-xL expression. These results indicate that SCL derived from RA synovial tissue play a role in promoting B cell survival by inducing Bcl-xL expression and blocking B cell apoptosis in a CD49d/CD29-CD106-dependent manner.

OBJECTIVE

This study examined the effects of acute psychological stress and exhaustive exercise on the expression and density of adhesion molecules (L-selectin, lymphocyte function antigen-1 [LFA-1], and intracellular adhesion molecule-1 [ICAM-1]) on monocytes, granulocytes, and lymphocytes.

METHODS

Forty-five healthy volunteers performed a 15-minute public speaking task and a 15- to 18-minute bicycle ergometer challenge.

RESULTS

In general, both the exercise and speaking tasks led to increases in the number of circulating leukocytes and lymphocyte subsets. The density of L-selectin (CD62L) on mixed lymphocytes and T lymphocytes was decreased in response to exercise (p values < .001). Both stressors led to an increased density of LFA-1 (CD11a) on mixed lymphocytes (p values < .01), whereas CD11a density on monocytes and granulocytes remained unchanged. ICAM-1 (CD54) density was unaffected, but the number of lymphocytes, monocytes, and granulocytes expressing CD54 increased in the circulation on both stressors.

CONCLUSIONS

The data indicate that both psychological stress and exercise have significant effects on cellular expression of adhesion molecules on circulating leukocytes. Given the crucial role that adhesion molecules on circulating cells play in inflammation and disease, these findings may have clinical relevance in sympathetic nervous system-induced immune activation.

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a receptor present on T cells that plays a critical role in the downregulation of antigen-activated immune responses. CTLA-4 interacts with the ligands CD80 and CD86 on antigen-presenting cells (APC), and also directs the assembly of inhibitory signalling complexes that lead to quiescence or anergy. In this study, we show that human monocytes constitutively express CTLA-4. About 3% of monocytes expressed CTLA-4 on the cell surface, whereas the intracellular expression was higher and present in about 20% of the monocytes. The sequences of the cDNAs from human monocytes were identical to the sequences of CTLA-4 from T cells. Expression of CTLA-4 was also confirmed in the activated myelomonocytic cell lines U937 and THP-1. Monocytes, but not T cells, activated by interferon (IFN)-gamma also secreted soluble CTLA-4 in vitro. The CTLA-4 expression was upregulated upon treatment with phorbol 12-myristate 13-acetate (PMA) and IFN-gamma. This increased expression could be partially abolished by staurosporine, an inhibitor of protein kinase C (PKC). Ligation of CTLA-4 in the monocyte-like cell-line U937 with antibodies against CTLA-4 partially inhibited the proliferation of cells and the upregulation of cell-surface markers CD86, CD54, HLA-DR and HLA-DQ induced by IFN-gamma and Staphylococcus aureus, Cowan I strain (SAC). Ligation of CTLA-4 suppressed the PMA-stimulated activation of transcription activator protein 1 (AP-1) and nuclear factor (NF)-kappaB in the U937 cell line, indicating the involvement of an inhibitory signal transduction. These data provide the first evidence that CTLA-4 is constitutively expressed by monocytes and thus might be important for the regulation of immune mechanisms associated with monocytes.

Murine bone marrow-derived dendritic cells (DC) can phagocytose and process Salmonella enterica serovar Typhimurium for peptide presentation on major histocompatibility complex class I (MHC-I) and MHC-II molecules. To investigate if a serovar Typhimurium encounter with DC induces maturation and downregulates their ability to present antigens from subsequently encountered bacteria, DC were pulsed with serovar Typhimurium 24 h prior to coincubating with Escherichia coli expressing the model antigen Crl-OVA. Quantitating presentation of OVA epitopes contained within Crl-OVA showed that Salmonella-pulsed DC had a reduced capacity to process Crl-OVA-expressing E. coli for OVA(257-264)/K(b) and OVA(265-277)/I-A(b) presentation. In addition, time course studies of DC pulsed with Crl-OVA-expressing serovar Typhimurium showed that OVA(257-264)/K(b) complexes could stimulate CD8OVA T-hybridoma cells for <24 h following a bacterial pulse, while OVA(265-277)/I-A(b) complexes could stimulate OT4H T-hybridoma cells for >24 but <48 h. The phoP-phoQ virulence locus of serovar Typhimurium also influenced the ability of DC to process Crl-OVA-expressing serovar Typhimurium for OVA(265-277)/I-A(b) presentation but not for OVA(257-264)/K(b) presentation. Furthermore, pulsing of DC with serovar Typhimurium followed by incubation for 24 or 48 h altered surface expression of MHC-I, MHC-II, CD40, CD54, CD80, and CD86, generating a DC population with a uniform, high expression level of these molecules. Finally, neither the serovar Typhimurium phoP-phoQ locus nor lipopolysaccharides (LPS) containing lipid A modifications purified from phoP mutant strains had a different effect on DC maturation from that of wild-type serovar Typhimurium or purified wild-type LPS. Thus, these data show that Salmonella or Salmonella LPS induces maturation of DC and that this process is not altered by the Salmonella phoP virulence locus. However, phoP did influence OVA(265-277)/I-A(b) presentation by DC infected with Crl-OVA-expressing serovar Typhimurium when quantitated after 2 h of bacterial infection.

Dendritic cells (DC) play a central role in immunity/tolerance decision, depending on their activation/maturation state. TNF-alpha is largely produced in the skin under inflammatory conditions. However, it still remains to be defined how TNF-alpha modulates the activation status of human LC, the most specialized DC controlling skin immunity. Here, we reported that fresh immature LC, highly purified from healthy human skin and exposed for two days to TNF-alpha under serum-free conditions, expressed up-regulated level of co-stimulatory molecules (CD40, CD54, CD86), maturation markers (CD83, DC-LAMP), CCR7 lymph node homing receptor, and down-regulated Langerin level, in a dose-dependent manner. This mature phenotype is closely associated with enhanced LC allostimulatory capacity. Furthermore, TNF-alpha significantly increased the number of viable LC and decreased their spontaneous apoptosis. More importantly, TNF-alpha induced LC to produce both IFN-gamma-inducible-protein IP-10/CXCL10, a Th1-attracting chemokine and IL-12 p40. Bioactive IL-12 p70 was never detected, even after additional CD40 stimulus. The results implicate LC as an effective target through which TNF-alpha may up- or down-regulate the inflammatory skin reactions.

CD80(B7-1) and CD86(B7-2) co-stimulatory molecules have been reported to activate Th1/Th2 development pathways differentially. It is well known that Langerhans cells (LC), potent antigen-presenting dendritic cells in the epidermis, express several co-stimulatory molecules and that this expression is modulated by several cytokines. Based on the recently reported effect of interferon (IFN)-gamma and interleukin (IL-)-10 on the expression of CD80 and CD86 by LC, we examined the effects of these cytokines on the expression of CD54 (intercellular adhesion molecule-1) and CD40 in addition to CD80 and CD86 on LC, and correlated the expression of each co-stimulatory molecule with antigen presentation for a Th1 clone by cultured LC (cLC) treated with these cytokines. LC cultured for 72 h significantly up-regulated MHC class II antigen expression and all the co-stimulatory molecules were examined. As previously reported, IL-10 or IFN-gamma inhibited the up-regulation of CD80 expression. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) partially restored the suppression of CD80 expression induced by IFN-gamma on cultured LC, while it had virtually no effect on the inhibition induced by IL-10. Antigen presentation for the myoglobin-specific syngeneic Th1 clone by cLC, which were pre-incubated with these cytokines, correlated well with their CD80 expression. In addition, among the antibodies for CD80, CD86, CD28 or CD40, the suppression of the Th1 clone stimulation by LC was found to occur only with anti-CD80 and anti-CD28 antibodies. Finally, we studied the effects of IFN-gamma and IL-10 on GM-CSF production by epidermal keratinocytes (KC). We could show that only IFN-gamma, but not IL-10, suppressed GM-CSF production by KC. These findings suggest that both IFN-gamma and IL-10 suppress antigen presentation by LC for Th1 cells by suppressing their CD80 expression. The inhibitory effect of IFN-gamma on CD80 expression on LC appears to be partially mediated through the suppression of GM-CSF production by KC.

Persistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered atwww.clinicaltrials.gov as #NCT01237249.

The mechanisms that underlie the potent Th1-adjuvant capacity of poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (NPs) were investigated. Traditionally, polymer NPs have been considered delivery systems that promote a closer interaction between antigen and antigen-presenting cells (APCs). Our results revealed that poly(anhydride) NPs also act as agonists of various Toll-like receptors (TLRs) (TLR2, -4, and -5), triggering a Th1-profile cytokine release (gamma interferon [IFN-gamma], 478 pg/ml versus 39.6 pg/ml from negative control; interleukin-12 [IL-12], 40 pg/ml versus 7.2 pg/ml from negative control) and, after incubation with dendritic cells, inducing a 2.5- to 3.5-fold increase of CD54 and CD86 costimulatory molecule expression. Furthermore, in vivo studies suggest that NPs actively elicit a CD8(+) T-cell response. Immunization with empty NPs resulted in a significant delay in the mean survival date (from day 7 until day 23 postchallenge) and a protection level of 30% after challenge against a lethal dose of Salmonella enterica serovar Enteritidis. Taken together, our results provide a better understanding of how NPs act as active Th1 adjuvants in immunoprophylaxis and immunotherapy through TLR exploitation.

Acinetobacter baumannii is an increasing hospital-acquired pathogen that causes a various type of infections, but little is known about the protective immune response to this microorganism. Outer membrane protein A of A. baumannii (AbOmpA) is a major porin protein and plays an important role in pathogenesis. We analyzed interaction between AbOmpA and dendritic cells (DCs) to characterize the role of this protein in promoting innate and adaptive immune responses. AbOmpA functionally activates bone marrow-derived DCs by augmenting expression of the surface markers, CD40, CD54, B7 family (CD80 and CD86) and major histocompatibility complex class I and II. AbOmpA induces production of Th1-promoting interleukin-12 from DCs and augments the syngeneic and allogeneic immunostimulatory capacity of DCs. AbOmpA stimulates production of interferon-gamma from T cells in mixed lymphocyte reactions, which suggesting Th1-polarizing capacity. CD4(+) T cells stimulated by AbOmpA-stimulated DCs show a Th1-polarizing cytokine profile. The expression of surface markers on DCs is mediated by both mitogen-activated protein kinases and NF-kappaB pathways. Our findings suggest that AbOmpA induces maturation of DCs and drives Th1 polarization, which are important properties for determining the nature of immune response against A. baumannii.

OBJECTIVE

This phase I study aimed to establish the biologic effects and MTD of the agonistic IgG1 chimeric anti-CD40 antibody ChiLob7/4 in patients (pts) with a range of CD40-expressing solid tumors and diffuse large B-cell lymphoma, resistant to conventional therapy. Potential mechanisms of action for agonistic anti-CD40 include direct cytotoxic effects on tumor cells and conditioning of antigen-presenting cells.

METHODS

ChiLob7/4 was given by IV infusion weekly for 4 doses at a range from 0.5 to 240 mg/dose. Validated ELISAs were used to quantify ChiLob7/4 in serum and test for anti-chimeric MAb (HACA) responses. Pharmacodynamic assessments included quantitation of T-cell, natural killer-cell, and B-cell numbers and activation in blood by flow cytometry and a panel of cytokines in plasma by Luminex technology. Planned dose escalation was in cohorts of 3 patients until MTD or biologic effect, defined as reduction of peripheral blood CD19(+) B cells to 10% or less of baseline.

RESULTS

Twenty-nine courses of treatment were given to 28 subjects. The MTD was 200 mg × 4, with dose-limiting toxicity of liver transaminase elevations at 240 mg. At 200 mg (range between 2.1 mg/kg and 3.3 mg/kg based on patient body weight), the trough level pretreatment was above 25 μg/mL. Grade 1-2 infusion reactions were seen above the dose of 16 mg, but could be prevented with single-dose corticosteroid premedication. HACA responses were seen after doses between 1.6 mg and 50 mg, but not above this. There were dose-dependent falls in blood B-cell numbers accompanied by reduced expression of CD21, and transient reductions in NK cell numbers with increased CD54 expression from 50 mg upward. MIP-1β and IL12 plasma concentrations rose after doses above 16 mg. Fifteen of 29 treatments were accompanied by disease stabilization for a median 6 months, the longest for 37 months.

CONCLUSIONS

ChiLob7/4 can activate B and NK cells at doses that can be administered safely, and should be tested in combination with other antibodies and chemotherapy agents.

The expression and co-expression profiles of functionally important monocyte surface markers were compared between control and HIV+ individuals using combined physical gating and dim CD4 expression to delineate the monocytes. The Fc gamma RII (CD32), the MHC class II antigen HLA-DR and the adhesion molecules CD11a (LFA-1 alpha), CD18 and CD54 (ICAM-1) showed an unimodal distribution. Of these markers, CD11a and HLA-DR were up-regulated in the HIV+ subjects compared with controls. The expression levels of the adhesion molecules correlated with each other in both patients and controls. The CD11b (CR3-alpha), CD14, Fc gamma RI, and Fc gamma RIII markers were bimodally distributed. Compared with controls, monocytes from seropositives contained fewer CD14bright+ cells, an equal proportion of Fc gamma RIbright+ cells, but twice as many Fc gamma RIII+ cells. The expression level of Fc gamma RI and CD11b within their brightly positive subset increased as CD4 T cells decreased. Both in patients and controls, co-expression of bright CD11b, CD14 and Fc gamma RI was shown, whereas the Fc gamma RIII+ cells were negative or dim positive for the former triad. We conclude that the expression of two Fc gamma R (I and III), of the adhesion molecules CD11a and CD11b and of HLA-DR showed particular alterations on monocytes from HIV+ subjects. The relationship of these phenotypic observations with altered cytokine profiles and altered monocyte function is discussed.

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1alpha, IL-1beta, IL-6, and TNF-alpha, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.

Lung dendritic cells (DCs) from mice were enriched to 92-99% purity using a multistep enrichment protocol which included fluorescence-activated cell sorting. DCs were analyzed for expression of cell surface molecules, function in a mixed leukocyte reaction (MLR), and dependence on accessory molecules in stimulating an MLR. DCs possessed potent accessory properties in vitro, while interstitial macrophages (IM), which are Ia-negative, displayed little MLR-stimulating function of their own. However, IM were capable of enhancing DC-initiated T cell proliferation via a cell contact mechanism. These results indicated that murine lung DCs functioned as stimulators of primary T cell responses, and that cells in the local environment influenced their function. Lung DCs expressed surface molecules typical of DCs from other sites including CD11a, CD54, CD80, and CD86. As is true for DCs in other sites, costimulatory molecules including CD80, CD86, CD40L, CD2, CD54, and CD11a played important roles in lung DC-initiated T cell proliferation. Interestingly, anti-CD86 monoclonal antibody (mAb) had little inhibitory effect on the MLR unless it was added in combination with anti-CD80 mAb. These studies suggest that CD80 on lung DCs can provide a costimulatory signal to allogeneic T cells in the absence of CD86 signaling, but that CD86 functions poorly except when CD80 is also engaged.

The adjuvanticity of MALP-2, a 2-kDa synthetic lipopeptide with macrophage-stimulatory activity, was evaluated in BALB/c mice using beta-galactosidase (beta-gal) as model antigen. When co-administered with beta-gal by either the intranasal (i.n.) or i.p. route, MALP-2 (0.5 microg) was capable of increasing beta-gal-specific serum IgG titers by 675-3,560-fold (i.n.) and 64-128-fold (i.p.), respectively, as compared to immunization with beta-gal alone. Using MALP-2, almost maximal IgG responses were already stimulated following the first immunization, and the IgG titers were similar to those observed using 10 microg of cholera toxin B subunit (CTB) as adjuvant. The mucosal immune system was also effectively stimulated (p<0.05) when MALP-2 was administered by the i.n. route (36% and 23% of beta-gal-specific IgA in lung and vaginal lavages, respectively). The i.n. co-administration of MALP-2 stimulated a stronger cellular immune response than CTB, both in submandibular lymph nodes and spleen (p<0.05). The analysis of beta-gal-specific IgG isotypes and the profiles of cytokines secreted by in vitro re-stimulated cells showed that co-administration of MALP-2 triggered a dominant Th2-response pattern. A recruitment of B220(+) and MAC-1(+) cells with an up-regulated expression of MHC class I, CD80 (B7.1) and CD54 (ICAM-1) was observed in nasal associated lymphoid tissues from MALP-2 treated mice. Taken together, our results demonstrated that the synthetic lipopeptide MALP-2 represents a very promising adjuvant for the mucosal delivery of vaccine antigens.

Mesenchymal stem cells (MSC) can be isolated from many sites adults and the fetus. Cells with osteoblastic, chondrogenic, leiomiogenic and stromogenic potentials have been obtained from the bovine artery wall, and we now show that MSC can be isolated also from the adult human vein wall. Cells detached from internal surface of the saphenous vein are cultured in vitro for 2-3 weeks and replated weekly. The culture forms a semi-confluent layer of spindle-shaped cells that are CD13(+), CD29(+), CD44(+), CD34(-), CD45(-), CD14(-), CD133(-), CD31(-), CD33(-), CD54(+), CD106(-), CD90(+), KDR(-), cadherin-5-, HLA class I(+) and HLA-DR- and differentiate in vitro into osteoblasts, chondrocytes and adipocytes. Gene expression, when compared with seven other normal tissues, shows strong similarity with MSC obtained from other sources. Three genes more expressed in saphenous MSC than in the other two MSC are related to angiogenesis, and the expression of two of them is shared by endothelial cells. These results demonstrate that the human vein wall contains mesenchymal cells with morphologic features, immunophenotypic markers, gene expression profile and differentiation potential that are similar to MSC obtained from the bone marrow and from the umbilical vein.

Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-specific T cells in melanoma patients. However, it is not known whether autologous DCs, differentiated by two of the most efficient protocols (from CD34+ progenitors or from monocytes), are equally effective as professional antigen-presenting cells (APCs) when the patients have a low frequency of peptide-specific precursors. To this end, a limiting dilution assay was applied to evaluate the frequency of antigen-specific CTL precursors (CTLps) in peripheral blood of HLA-A*0201+ melanoma patients. Then, from two melanoma patients showing low frequency of CTLps to melanoma antigen-A/melanoma antigen recognized by T cell (Melan-A/Mart-1)(27-35) peptide, autologous DCs were differentiated from granulocyte colony-stimulating factor-mobilized CD34+ progenitors or from monocytes. CD34+- and monocyte-derived DCs were characterized by a similar proportion of CD1a+ cells expressing HLA class II antigens and CD54, CD80, and CD86 molecules. Both types of DC presented Melan-A/Mart-1(27-35) and tyrosinase(369-377) peptides to melanoma-specific CTL clones and were equally effective as peptide-pulsed APCs in the activation of influenza A matrix(58-66)-specific CTLs from high-frequency precursors (1294/10(6) and 1789/10(6) lymphocytes in the two patients). However, efficient activation of Melan-A/Mart-1(27-35)-specific CTLs from low-frequency precursors (158/10(6) and 77/10(6) lymphocytes) of the two patients was markedly dependent on the use of peptide-loaded CD34+-derived DCs. These results suggest that CD34+- and monocyte-derived DCs are not functionally equivalent APCs for the activation of low-frequency peptide-specific CTLps.

LeIF, a gene homologue of the eukaryotic initiation factor 4A was first described as a leishmanial antigen that induced a Th1-type T cell response in peripheral blood mononuclear cells (PBMC) from leishmaniasis patients. Moreover, the interferon (IFN)-gamma production by PBMC was found to be interleukin (IL)-12 dependent. Herein, we characterize the effects of LeIF on cytokine production and expression of surface molecules by normal human monocytes as well as by monocyte-derived macrophages and dendritic cells (MoDC). LeIF was a strong inducer of IL-12 and, to a lesser extent, of IL-10 and tumor necrosis factor (TNF)-alpha in macrophages and MoDC. IL-12 production did not require CD40 triggering, confirming that the ability of LeIF to induce IL-12 was not mediated through an effect on T cells. However, addition of soluble CD40 ligand (L) synergistically augmented IL-12 production in macrophages and MoDC. The cytokine-inducing activity of LeIF is located in the N-terminal portion of the molecule and was both proteinase K sensitive and polymyxin B resistant. LeIF, lipopolysaccharide and fixed Staphylococcus aureus all induced comparable amounts of IL-12, validating the potent cytokine-inducing effects of LeIF. Moreover, of these stimuli, LeIF had the highest IL-12/IL-10 and IL-12/TNF-alpha ratio demonstrating the preference of LeIF for IL-12 induction. Studies investigating the expression of surface molecules showed that LeIF up-regulated B7-1 and CD54 (ICAM-1) on macrophages and MoDC. To our knowledge this is the first report describing IL-12 production, up-regulation of co-stimulatory and intercellular adhesion molecules by monocytic antigen-presenting cells in response to a protein from a pathogenic microorganism. These immunomodulatory characteristics of LeIF might be excellent properties for a Th1-type adjuvant.

Cytokines secreted by macrophages play important roles in the immune response to Trypanosoma cruzi. Here, we report that a purified glycosylphosphatidylinositol (GPI)-anchored mucin from the T. cruzi membrane, Ag C10, is able to bind to the macrophage cell surface and blocks the subsequent binding of mAb against CD62L/L-selectin, whereas binding of mAbs directed against other monocyte surface molecules is unaffected. In addition, Ag C10 binding to macrophages triggered a CD54/ICAM-1-mediated cell adhesion as well as an increase in intracellular Ca2+, which was further augmented by cross-linking the Ag C10-bound surface receptors by mAb against Ag C10. Interestingly, Ag C10-treated monocytes secreted IL-1beta, but not TNF-alpha or IL-12. Moreover, these cells could secrete IL-1beta, but not TNF-alpha or IL-12, after activation with LPS. T. cruzi-infected macrophages displayed similar alterations in cytokine secretion, with an impaired ability to secrete IL-12 and TNF-alpha, but not IL-1beta, upon LPS activation. These effects were substantially inhibited by neutralizing mAb against Ag C10. These effects appeared to take place at the transcriptional level, since mRNA for TNF-alpha, but not that for IL-1beta, was drastically reduced in LPS-stimulated infected cells treated with Ag C10. Conceivably, inhibition of TNF-alpha and IL-12 by T. cruzi could be involved in the evasion of the immune response by this parasite.

APO-1 is a 48-Kd transmembrane glycoprotein identical to the Fas antigen and belongs to the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor family of surface molecules. Cross-linking of APO-1 induces apoptotic cell death in sensitive cells. We show here that APO-1 is an activation molecule on B cells. It was induced/enhanced on dense and buoyant tonsillar B cells, respectively, through surface Ig cross-linking in combination with interleukin-2 or by interferon-gamma together with tumor necrosis factor-alpha. These conditions also increased the amount of intercellular adhesion molecule-1 (CD54) on these cells. Epstein-Barr virus transformants of peripheral B cells coexpressed APO-1 and CD54 at very high levels. Immunohistologically, Apo-1 was detectable at low levels in a subpopulation of follicle center B blasts and, at higher levels, in sinusoidal B cells. APO-1 was undetectable in follicular mantle B cells and plasma cells. In isolated tonsillar B cells, APO-1 was expressed in CD10+ follicle center cells. In acute B lymphoblastic leukemia, chronic B lymphocytic leukemia, and Burkitt's lymphomas, APO-1 and CD54 molecules were immunohistochemically undetectable. Coordinate expression of these antigens was found in mediastinal B-cell lymphomas. The mode of APO-1 and CD54 expression was correlated in follicle center cell lymphomas (P < .0019), but less stringently in hairy cell leukemia. No association was found in plasmacytomas. This was in line with the differential expression of these molecules found in reactive plasma cells. Expression of APO-1 in B cells of different stages of differentiation and, correspondingly, in certain B-cell neoplasias might suggest a role of this molecule in the induction of B-cell apoptosis. This function might be influenced by CD54 and CD54-mediated signals.

Overexpression of p185(c-erbB2) (p185/NEU/HER2) by tumor cells is associated with a poor prognosis in many but not all studies of breast and ovarian cancer. The poor prognosis associated with overexpression of p185(c-erbB2) could result from an increased growth rate or increased invasive potential. The p185(c-erbB2) tyrosine kinase receptor can be activated with agonistic antibodies directed against p185(c-erbB2) or with the ligand heregulin through a combinatorial interaction with erbB3 or erbB4. Consequently, we have asked whether heregulin or agonistic antibodies increase anchorage-independent growth or invasiveness of the SKBr3 breast cancer cell line, which overexpresses p185(c-erbB2). Incubation of SKBr3 breast cancer cells with heregulin inhibited anchorage-independent growth while enhancing tyrosine phosphorylation of p185(c-erbB2). Heregulin treatment also increased adhesion of SKBr3 cells to plastic and increased invasiveness of tumor cells into Matrigel membranes while increasing expression of the CD44 (HCAM) and CD54 (ICAM-1) adhesion molecules. Tumor cell invasion of Matrigel membranes was partially blocked by either anti-CD44 or anti-CD54 antibodies, indicating a role for these adhesion molecules in the invasion process. Compatible with the increased invasiveness, heregulin increased expression of the matrix metalloproteinase 9. In contrast, the agonistic anti-p185(c-erbB2) antibody ID5 induced only a subset of the responses induced by heregulin. ID5 induced tyrosine phosphorylation of p185(c-erbB2), increased invasiveness, and increased expression of CD44. Despite the similarity of effects of ID5 and heregulin on some outcomes, the ID5 antibody failed to increase adhesion to plastic, expression of CD54, or production of matrix metalloproteinase 9. Thus, the ID5 agonistic anti-p185(c-erbB2) antibody mimics rather than antagonizes some but not all of the actions of heregulin. Moreover, the poor prognosis of breast and ovarian cancers that overexpress p185(c-erbB2) could relate in part to enhanced invasiveness rather than to increased proliferative capacity.

Human mesenchymal stem cells (MSCs) have the potential to differentiate into cells of connective tissue lineages, including bone, cartilage, fat, muscle and also neurons. In our study we have examined the phenotypic profile of human adipose tissue-derived stem cells (hASCs) and compared different osteogenic-inductive media to assess hASC differentiation. Cells were enzymatically isolated from adipose tissues derived by liposuction from several adult human donors, purified and then expanded in culture. We obtained an abundant yield of hASCs with a constant proliferative trend, a doubling time of about 68 h and a mild variable clonogenic capacity. At passage 4, hASCs expressed MSC-related cell surface antigens (CD13, CD105, CD54, CD90, CD44), and subsequently hASCs were induced to differentiate into the osteogenic lineage for at least 3 weeks of culture in two distinct media, OM1 and OM2, differing in dexamethasone and ascorbic acid concentrations. Osteogenic differentiation of OM1- and OM2-cultured cells was assessed by evaluating cell morphology, osteopontin expression, alkaline phosphatase activity and calcium deposition. OM2 medium showed a higher osteogenic potential than OM1, as assessed by increased levels of calcium deposition, alkaline phospatase activity and osteopontin expression in comparison with OM1-differentiated cells. We conclude that hASCs efficiently differentiate into osteogenic lineage, particularly when cultured in inductive medium supplemented with 10 nM dexamethasone and 150 microM ascorbic acid.

Activation of resting human CD4+ T cells mediated by mAb ligation of the TCR/CD3 complex requires costimulatory signals to result in proliferation; these can be provided by intercellular cell adhesion molecule-1 (ICAM-1, CD54) a natural ligand of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18). We analyzed early signaling events involved in T cell activation to determine the contribution by the LFA-1/ICAM-1 interaction. We studied in detail the hydrolysis of phosphatidylinositol(4,5)bisphosphate and intracellular levels of free Ca2+ during stimulation with beads coated with the CD3 mAb OKT3 alone or in combination with purified ICAM-1 protein. Our investigations show no response to LFA-1/ICAM-1 alone, but that costimulation by LFA-1/CAM-1 interaction induces prolonged inositol phospholipid hydrolysis (up to 4 h), resulting in generation of both inositol(1,4,5)phosphate3 and inositol(1,3,4,5)phosphate4 and their derivatives. Based on studies with cycloheximide, this costimulatory effect of prolonged inositol phospholipid hydrolysis appears dependent in part on de novo protein synthesis. A sustained increase in intracellular levels of free Ca2+ level is also observed after LFA-1/ICAM-1 costimulation, which is at least partly dependent on extracellular sources of Ca2+. Kinetic studies indicate that costimulation requires a minimal period of 4 h of LFA-1/ICAM-1 interaction to provide maximal costimulation for OKT3-mediated T cell proliferation. Thus, the necessary costimulation required for OKT3-mediated proliferation in this model system may be provided by an extended LFA-1/ICAM-1 interaction that in combination with OKT3 mAb leads to signal-transducing events, resulting in prolonged phospholipase C activation and phosphatidylinositol(4,5)bisphosphate hydrolysis, and a sustained increase in intracellular levels of free Ca2+.

We compared immunologic functions of human vascular smooth muscle cells (VSMC) with those of endothelial cells (EC) cultured from saphenous vein. Both cell types can express comparable levels of MHC class II molecules. However, class II-positive VSMCs, unlike ECs, do not stimulate resting CD4+ T cell proliferation. Limiting dilution analyses revealed IL-2-producing cells alloreactive to class II-positive ECs but not VSMCs. Class II molecules on VSMCs are functional, inducing CD25 expression on resting CD4+ T cells and stimulating proliferation of CD4+ T cells that have been pre-activated by ECs. VSMC expression of the co-stimulator molecules CD44, CD54, CD58, and CD59 is comparable to EC expression, and neither VCAM-1 nor B7 are expressed on either cell. However, VSMCs are less efficient than ECs at co-stimulating IL-2 production by PHA-stimulated PBL. VSMCs but not ECs cultured across a Transwell inhibit CD4+ T cell proliferation to allogeneic ECs and, to a lesser extent, IL-2 production in the same assay. Inhibition of proliferation cannot be transferred by VSMC-conditioned media, nor reversed by inhibitors of prostaglandin synthesis, TGF-beta 1, or nitric oxide synthesis. CD4+ T cells cocultured with class II-positive VSMCs proliferate to a subsequent challenge with ECs from the same donor as well as freshly isolated T cells. We conclude that VSMCs express functional MHC class II molecules and stimulate pre-activated T cells. However, VSMCs lack adequate costimulators to fully stimulate resting T cells, and VSMCs inhibit T cell proliferation.

Caveolae are plasma membrane invaginations that function as a center for signal transduction. Recent studies indicate that caveolins, the main proteins in caveolae, serve as scaffolding proteins onto which many classes of signaling molecules are assembled. There are multiple forms of caveolins: caveolin-1 and caveolin-2 are expressed as stable heterooligomeric complexes within most cell types, while caveolin-3 is restricted to striated muscle cells. However, neither caveolin proteins nor caveolae structures are detected in peripheral blood cells or blood cell lines. We identified caveolin-1 in one T cell leukemia cell line, a subline of Jurkat cells, by immunostaining and Western blotting. The cells showed enlarged cell bodies similar to activated T cells. This led us to investigate caveolin expression in adult T cell leukemia (ATL) cell lines, which are known to be constitutively activated. Two of five ATL cell lines expressed caveolin-1. The phenotype of caveolin-1-positive cells expressed not only high levels of the T cell activation markers, as with CD25 or HLA-DR, but also CD54 at extremely high levels. These findings demonstrate for the first time that hematological cells express caveolin-1 in certain states of cell activation. In addition, the caveolin-1 expression may be a useful marker for the diagnosis of ATL malignancy.