Microglia cells are the principal immune effector elements of the brain responding to any pathological event. To elucidate the possible role of microglia in initial non-inflammatory demyelination in canine distemper virus (CDV) infection, microglia from experimentally CDV infected dogs were isolated ex vivo by density gradient centrifugation and characterized immunophenotypically and functionally using flow cytometry. Results from dogs with demyelinating lesions were compared to results from recovered dogs and two healthy controls. CDV antigen could be detected in microglia of dogs with histopathologically confirmed demyelination. Microglia of these dogs showed marked upregulation of the surface molecules CD18, CD11b, CD11c, CD1c, MHC class I and MHC class II and a tendency for increased expression intensity of ICAM-1 (CD54), B7-1 (CD80), B7-2 (CD86), whereas no increased expression was found for CD44 and CD45. Functionally, microglia exhibited distinctly enhanced phagocytosis and generation of reactive oxygen species (ROS). It was concluded that in CDV infection, there is a clear association between microglial activation and demyelination. This strongly suggests that microglia contribute to acute myelin destruction in distemper.

Asthma is a chronic inflammatory disease process involving the conductive airways of the human lung. The dysregulated inflammatory response in this disease process may involve multiple cell-cell interactions mediated by signaling molecules, including lipid mediators. Extracellular vesicles (EVs) are lipid membrane particles that are now recognized as critical mediators of cell-cell communication. Here, we compared the lipid composition and presence of specific lipid mediators in airway EVs purified from the bronchoalveolar lavage (BAL) fluid of healthy controls and asthmatic subjects with and without second-hand smoke (SHS) exposure. Airway exosome concentrations were increased in asthmatics, and correlated with blood eosinophilia and serum IgE levels. Frequencies of HLA-DR+ and CD54+ exosomes were also significantly higher in asthmatics. Lipidomics analysis revealed that phosphatidylglycerol, ceramide-phosphates, and ceramides were significantly reduced in exosomes from asthmatics compared to the non-exposed control groups. Sphingomyelin 34:1 was more abundant in exosomes of SHS-exposed asthmatics compared to healthy controls. Our results suggest that chronic airway inflammation may be driven by alterations in the composition of lipid mediators within airway EVs of human subjects with asthma.

Extranodal, nasal NK/T-cell lymphomas are regularly Epstein-Barr virus (EBV)-positive, with a type II latency pattern, expressing thus EBNA-1 and LMP1. The contribution of EBV to the tumor development is not known. Similarly to normal natural killer (NK) cells, cell lines derived from malignancies with a NK phenotype require IL-2 for in vitro proliferation. In our effort to explore the contribution of EBV, particularly the role of the LMP1 protein, to the pathogenesis of the NK lymphoma we found that its expression, studied in the NK-lines SNK6 and KAI3, depended on the supply of IL-2 or other cytokines. In the absence of IL-2 other cytokines, such as IL-10 and IFN-gamma, could maintain LMP1, but the cells did not proliferate. When grown in IL-2, the SNK6 cells produced IL-10 and IFN-gamma, and these cytokines mediated the expression of LMP1. IL-10 treatment enhanced, while IFN-gamma receptor blocking antibody reduced, the expression of CD25 and CD54 in the EBV-positive, but not in the EBV-negative lines. IL-10 treated cells required lower amount of IL-2 for proliferation compared to the untreated cells. This effect was seen only with the EBV-positive NK lines in which LMP1 and CD25 were concomitantly upregulated. By this mechanism EBV could have an important role in the development of NK lymphoma since the inflammatory component in the tumor tissue can provide these cytokines.

Although vascular pathologies such as vasculitis, endocarditis and mycotic aneurysms have been described in brucellosis patients, the interaction of Brucella with the endothelium has not been characterized. In this study we show that Brucella abortus and Brucella suis can infect and replicate in primary human umbilical vein endothelial cells (HUVEC) and in the microvascular endothelial cell line HMEC-1. Infection led to an increased production of IL-8, MCP-1 and IL-6 in HUVEC and HMEC-1 cells, and an increased expression of adhesion molecules (CD54 in both cells, CD106 and CD62E in HUVEC). Experiments with purified antigens from the bacterial outer membrane revealed that lipoproteins (Omp19) but not lipopolysaccharide mediate these proinflammatory responses. Infection of polarized HMEC-1 cells resulted in an increased capacity of these cells to promote the transmigration of neutrophils from the apical to the basolateral side of the monolayer, and the same phenomenon was observed when the cells were stimulated with live bacteria from the basolateral side. Overall, these results suggest that Brucella spp. can infect and survive within endothelial cells, and can induce a proinflammatory response that might be involved in the vascular manifestations of brucellosis.

Epstein-Barr virus is associated with several human malignancies including Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's disease (HD). To examine the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1) in the pathogenesis of HD, we transfected the gene into the HD cell line L428. EBNA-1 expression was associated with significantly enhanced CD25 expression (interleukin 2 [IL-2]-receptor alpha chain) in transient and stably transfected L428 cells but did not affect the expression of IL-2 receptor beta and gamma chains. There was no up-regulation of the B-cell activation molecules CD23, CD30, CD39, CD40, CD44, CD71, and CD54 (intercellular adhesion molecule 1) or enhanced production of IL-6, IL-10, lymphotoxin alpha, and the soluble form of CD25. Stable EBNA-1-expressing L428 cells were nontumorigenic in SCID mice but showed enhanced lymphoma development in nonobese diabetic-SCID mice compared to mock-transfected cells.

The role of Langerhans cells (LC) in the initiation of an immune response to a viral infection remains unclear. In vivo epidermal infection with the arboviruses West Nile virus and Semliki Forest virus significantly increased the expression of major histocompatibility complex class II antigens, CD54, and CD80 on LC. Thus, during an epidermally acquired viral infection, local LC appear to mature to a phenotype approximating that of lymphoid dendritic cells. This change may be important in the activation of naïve T cells and the subsequent clearance of viral infection.

Age-related changes of immune functions have been extensively investigated in both humans and animal models; nevertheless, the literature on potential alterations of dendritic cells, potent antigen presenting cells responsible for initiating immune responses, with aging is very scarce. We studied the immuno-phenotype of peripheral blood dendritic cells of elderly and young subjects by three-color flow cytometry. In addition, the capacity of transendothelial migration, an important step in inflammatory reactions, of peripheral blood dendritic cells of elderly subjects was investigated in an in vitro model. The expression of HLA-DR in the peripheral blood dendritic cells of the elderly subjects was significantly decreased when compared to the young control subjects. The expression of various other surface markers was similar in the young and elderly subjects. The ability of transendothelial migration of dendritic cells was found to be unimpaired in the elderly subjects. Both in the young and elderly subjects a significantly higher expression of CD29, CD86, HLA-DR, and HLA-DQ in the dendritic cells that had migrated through the endothelium in comparison to nonadherent, nonmigrating cells was found. In the migrating dendritic cells of the elderly subjects a significantly increased expression of CD11c was observed, whereas the expression of CD54 was significantly enhanced in the migrating dendritic cells of the young subjects only. In conclusion, our results demonstrate intact functions and a normal immunophenotype of dendritic cells derived from elderly subjects. Dendritic cells thus seem to be functional and therefore are not responsible for the well-known decline of T cell functions with aging.

Airway macrophages are activated in asthmatic subjects. Peripheral blood monocytes from these subjects present some functional features of activation, but their membrane markers are not known. Recently a new subtype of blood monocytes, CD14+/CD16+, has been identified which possesses the characteristics of tissue macrophages. A study was carried out on nine normal subjects and 11 untreated asthmatics having variable severities of the disease to examine the phenotypic characteristics of monocytes. CD14, CD16, HLA-DR, CD11a, CD11b, CD44 and CD54 were studied using double fluorescence flow cytometry since these antigens have been defined in the CD14+/CD16+ monocytes. The functional activation of monocytes was examined using the release of superoxide anion. The co-expression of CD14 and CD16 by monocytes in terms of percentage and mean fluorescence intensity was significantly higher in asthmatics (P < 0.002 and P < 0.0001, respectively, Mann-Whitney U-test). There was no difference for the other membrane markers between asthmatics and normal subjects. Superoxide anion release was significantly increased in asthmatic subjects (P < 0.01). This study shows that most blood monocytes of asthmatics are CD14+/CD16+ and are likely to present features of tissue macrophages.

Endothelial cells lining the vasculature participate in a variety of physiological processes. Following cell activation, functional changes are accompanied by changes in the surface structure (or phenotype) of these cells. Studies to date have tended to concentrate on selective changes induced with one or two surface molecules. The following study uses a different approach, having assessed potential changes to the endothelial cell surface using a large number >> 120) of previously untested monoclonal antibodies, and the cytokines TNF-alpha and gamma-IFN, as well as the proteolytic enzyme thrombin. Antibody representatives from all cluster of differentiation groups CD1 through to CD54 were assessed in these studies, which used human umbilical vein endothelial cells. In line with previous observations, antibodies within CD9, CD13, CD26, CD29, CD31, CD34, CD44, CD46, CD47, CD49, CD51 and CD54 gave significant and consistent reactivity using non-stimulated ('quiescent') endothelium. Using parallel cells differentially stimulated with TNF-alpha, gamma-IFN or thrombin, antibodies within CD1 through to CD15, CDw17 to CD19, CD21 to CD23, CD26, CD27, CD29, CD30, CD33 to CD35, CD37, CD38, CD40, CD43 to CD46, CD48, CD51 to CD53 failed to provide any consistent alteration to reactivity patterns compared to non-stimulated cells. There did, however, appear to be some activation induced changes using antibodies within the other CD groups (i.e. CD16, CD20, CD24, CD25, CD28, CD31, CD32, CD36, CD39, CD41, CD42, CD47, CD49, CD50 and CD54) which ranged from minor to significant in scope and magnitude.

An investigation of proliferation and activation events in subsets of human CD4+ cells, defined by their expression of CD45RA and CD45R0, is reported. A single-laser based assay for the study of multiple surface antigens and two-parameter cell cycle analysis was used for sorting of and subsequent analysis of proliferation in CD4+CD45RA+ CD45R0-, CD4+CD45RA-CD45R0+ subsets and phenotypically intermediate stages. After labelling with BrdUrd, cells were sorted with flow cytometry on the basis of light-scattering properties and staining with anti-CD45RA, anti-CD45R0, and anti-CD4 markers. Sorted cells were double stained with anti-BrdUrd-antibodies and PI, and the frequencies of proliferating cells were determined. After 48 h, the highest rate of proliferation was found among cells with a phenotype intermediate between CD4+ CD45RA+CD45R0- and CD4+CD45RA-CD45R0+. After 72 h of culture, the situation was changed insofar as the point of highest proliferation had shifted towards the CD4+CD45RA-CD45R0+ population. These findings were further corroborated by four-colour staining with anti-CD4, anti-CD45RA, anti-CD45R0, and Hoechst 33342. This indicates that the phenotype transition is accompanied by cell proliferation. The correlated temporal expression of antigens related to activation (HLA-DR, CD25, CD69, CD71) and cell adhesion (CD11a, CD54, L-selectin) in each of the different subsets was also investigated. All the activation markers CD25, CD69, and CD71 show a more heterogeneous pattern of expression among the CD4+ CD45RA-CD45R0+ cells than the CD4+ CD45RA+CD45R0- cells, indicating a subpopulation of CD4+CD45RA-CD45R0+ cells responding more slowly to the mitogenic stimulation.

Fumaric acid esters are thought to improve psoriasis by altering leukocyte, keratinocyte, and/or endothelial functions. To determine specificity, kinetics, and molecular mechanisms of different fumaric acid esters in their ability to inhibit endothelial cell activation, we analyzed CD62E and CD54 expression in endothelial cells in vivo and in vitro. In lesional skin of psoriatic patients, oral fumaric acid ester treatment resulted in a marked reduction of CD62E but not CD54 expression on dermal microvessels. Using human umbilical vein endothelial cells, dimethylfumarate almost completely inhibited tumor-necrosis-factor-induced CD62E, but not CD54 expression at concentrations < or = 70 microM, mimicking the situation in vivo. A 60 min dimethylfumarate preincubation was sufficient to block tumor-necrosis-factor-induced CD62E expression for up to 24 h. In contrast, equimolar concentrations of methylhydrogenfumarate, the hydrolysis product of dimethylfumarate, did not suppress tumor-necrosis-factor-induced CD62E expression. Likewise, all fumaric acid esters other than dimethylfumarate were ineffective. Using CD62E, NF-kappa B, or AP-1-responsive promoter constructs, dimethylfumarate inhibited tumor-necrosis-factor-induced activation of the CD62E and the NF-kappa B but not the AP-1 promoter construct. In summary, at a dose range < or = 70 microM, dimethylfumarate appeared to be a specific inhibitor of CD62E expression in an NF-kappa B-dependent manner.

A 2-kDa synthetic derivative of the macrophage-activating lipopeptide (MALP-2) from Mycoplasma fermentans is a potent inducer of monocytes/macrophages and improves the immunogenicity of antigens co-administered by systemic and mucosal routes. Dendritic cells (DC) are the most potent antigen-presenting cells, which are able to prime naive T cells in vivo. To elucidate the underlying mechanisms of MALP-2 adjuvanticity, we analyzed its activity on bone marrow-derived murine DC. In vitro stimulation of immature murine DC with MALP-2 resulted in the induction of maturation with up-regulated expression of MHC class II, costimulatory (CD80, CD86) and adhesion (CD40, CD54) molecules. MALP-2 also enhances the secretion of cytokines (IL-1alpha, IL-6 and IL-12), and increases DC stimulatory activity on naive and antigen-specific T cells. Further studies demonstrated that MALP-2 treatment of DC results in a dose-dependent shift from the protein pattern of proteasomes to immunoproteasomes (up-regulation of LMP2, LMP7 and MECL1), which correlates with an increased proteolytic activity. Thus, the adjuvanticity of MALP-2 can be mediated, at least in part, by the stimulation of DC maturation, which in turn leads to an improved antigen presentation. Therefore, MALP-2 is a promising molecule for the development of immune therapeutic or prophylactic interventions.

Plasma cells (PC) localize to discrete areas of secondary lymphoid tissue and bone marrow (BM). The positioning of PC in different sites is believed to be regulated by chemokines and adhesion molecules expressed by accessory cells in the lymphoid tissue microenvironment. However, the mechanisms responsible for the positioning of PC within the red pulp (RP) of human spleen have not been elucidated. Therefore, we examined the contribution of human splenic stromal cells to the migration and function of human PC. Splenic PC expressed the chemokine receptor CXCR4 and responded to its ligand CXCL12. In contrast, PC lacked CXCR5 and CCR7, and consequently exhibited minimal migration towards CXCL13 and CCL21. Splenic stromal cells proved to be a rich source of CXCL12, and could induce the migration of human B cells. Furthermore, they supported Ig production by splenic PC mainly by secreting IL-6. Lastly, a striking difference between splenic and BM PC was the constitutive expression of CD11a by only splenic PC. Notably, splenic stromal cells expressed high levels of CD54, the counter-structure of CD11a, and splenic PC were positioned adjacent to stromal cells in the RP. Thus, we propose that stromal cells attract PC to the RP and contribute to their retention and function through the combined expression of CXCL12, CD54 and IL-6.

Alveolar epithelial cells type II (AEC-II) express MHC class II on their surface, an important prerequisite for antigen presentation. However, accessory signals are required for an efficient T-cell activation. We therefore isolated AEC-II from tumor-free sections of human lungs obtained by lobectomy/pneumectomy and purified the cells by magnetic-activated cell sorting. Furthermore, we tested the expression of CD54, CD58, CD80, and CD86 on AEC-II and evaluated their accessory function (AF) in cell culture using a coculture of interleukin-2 (IL-2), releasing Jurkat cells and AEC-II. An increased AF is documented by an elevated IL-2 release and expressed as accessory index (AI). In 33 experiments the AF of AEC-II proved to be highly variable. AI ranged between 0.3 and 17.1 with a median of 1.4 (0.3-17.1). Forty-four percent (4-77) of the AEC-II expressed HLA-DR, 44% (12-89%) of the cells expressed CD58, and CD54 was expressed by 55% (16-89%). AEC-II also expressed CD80 and CD86 (38% [0-77%] and 40% [4-68%], respectively). Interestingly, AEC-II released high levels of TGF beta (1730 pg/mL [771-5876]) and the accessory index could be increased (approximately 2-fold) by the addition of neutralizing anti-TGF beta antibodies or radiation. Thus, type II alveolar cells express costimulatory molecules and are able to deliver costimulatory signals for T cells, providing evidence that AEC-II are able to act as antigen-presenting and immunoregulatory cells of the lung. Additionally, the accessory function of AEC-II is under the control of endogenously released TGF beta.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.

Pulmonary surfactant and its lipid components inhibit cell proliferation and cytokine expression. Surfactant protein A (SP-A) can stimulate these same functions. We assessed the impact of SP-A and surfactant lipids on the expression of the cell surface markers, CD14, CD54 (intercellular adhesion molecule-1), and CD11b, by the human monocytic cell line THP-1 using fluorescent antibody staining and fluorescence-activated cell sorting. Under basal conditions CD14 and CD54 were undetectable, and CD11b was expressed at low levels. Incubation of the cells in 1,25(OH)2D3 alone, or with low doses of surfactant lipids, increased CD14, CD54, and CD11b. Expression was increased further by SP-A. However, the SP-A-induced increases in cell markers were blocked by simultaneous treatment with lipid. The results suggest that the ability of the macrophage to participate in an inflammatory response is enhanced by SP-A alone or by surfactant containing a higher than normal proportion of SP-A. They further suggest that the addition of lipids results in a phenotype less prone to initiate an inflammatory reaction.

The aim of this study was to determine the strength of the association between the human immune response and body mass index (BMI) and whether differences exist in the effects of obesity on selected immune parameters between men and women. Two hundred ninety participants were divided into groups according to sex and BMI. Parameters CD3, CD4, CD8, CD16+56, CD19, HLADR, CD11b, CD11c, and CD54 were quantified. Leukocyte and differential counts were performed. We observed elevation with regard to the normal weight group in the parameters of white blood cells, neutrophils, monocytes, CD3, CD4, CD19, and CD11b for the whole study group. A decrease was observed in the expression of CD16+56. The effect of BMI on the immune system was much more apparent in women. BMI was correlated with the majority of the measured parameters, reflecting a strong association between BMI and the human immune system.

Ligation of CD40 on the surface of B cells induces multiple phenotypic effects, many of which are mimicked by the EBV latent membrane protein 1 (LMP1) through its interaction with downstream components of the CD40 signaling pathway. Because the effects of LMP1 have been most closely studied in human Burkitt Lymphoma (BL) cell lines retaining a tumor biopsy-like phenotype in vitro, we have examined the response of a panel of such lines to CD40 ligation. Two distinct patterns of response were observed that were unrelated to the surface level of CD40 or to the EBV genome status of the lines. Following exposure to either CD40-specific mAbs or the soluble trimeric ligand (sCD40L), high responder (HR) lines showed rapid aggregation, activation of NF-kappa B, up-regulation of cell surface markers ICAM-1/CD54 and Fas/CD95, and growth inhibition. Aggregation was seen at lower doses than those required to elicit the other effects. By contrast, low responder (LR) lines showed no detectable response to CD40 mAbs, while their responses to sCD40L were limited to activation of NF-kappa B and up-regulation of CD95 only. However, in transfection experiments, LMP1 uniformly induced the full spectrum of phenotypic effects in both HR and LR lines. We conclude that some BL cell lines show a highly restricted response to CD40 ligation but remain fully susceptible to LMP1.

During ontogeny, the skin is progressively populated by major histocompatibility complex class II-negative dendritic cell (DC) precursors that then mature into efficient antigen-presenting cells (APC). To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an envAKR-mycMH2 fusion gene. These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum-free medium was promoted by granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage-CSF. FSDC expressed strong surface-membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H-2d,b+, I-Ad,b+, CD54+, CD11b+, CD11c+, 2.4G2+, F4/80+, CD44+, 2F8+, ER-MP 12-, Sca-1+, Sca-2+, NLDC-145-, B7.2+, B7.1-, J11d-, B220-, Thy-1-, and CD3-. FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction, and markedly increased this function after treatment with GM-CSF, GM-CSF and interleukin (IL)-4 or interferon-gamma (IFN-gamma); in contrast, stem cell factor, IL-1 alpha and tumor necrosis factor-alpha had no effect. Preculture with IFN-gamma was required for presentation of haptens to primed T cells in vitro. However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays. Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice. The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC-like surface phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g. cytokines) to express their antigen presenting potential in vitro.

Expression of immune accessory molecules, such as CD80 (B7-1), on antigen-presenting cells governs whether such cells can activate antigen-specific T cells. As such, the factors that regulate the expression of these accessory molecules may determine whether presentation of antigen leads to immune activation or anergy. We previously reported that anti-CD3-activated T cells (Ta) can induce expression of CD80 and CD54 (ICAM-1) on human B cells through a contact-dependent signal delivered to the CD40 molecule via the CD40 ligand. Here, we demonstrate that another molecule in the CD40-ligand family, namely tumor necrosis factor-alpha (TNF-alpha), also plays a role in the Ta-mediated induction of CD80 or CD54 on human B cells. Neutralizing mAbs specific for TNF-alpha can inhibit B cell expression of CD80 or CD54 that is induced when B cells are cultured with Ta cells or in Ta-cell conditioned media. Moreover, soluble, recombinant TNF-alpha or TNF-beta can induce significant increases in B cell expression of CD80 and CD54. The phenotypic changes effected by TNF-alpha can be recapitulated by crosslinking CD120b (p75 TNF-receptor), but not CD120a (p55 TNF-receptor), with mAbs presented on Fc gamma RII (CD32)-expressing L cells. IL-4 augments the expression of CD80 induced by crosslinking either CD40 or CD120b. However, although IL-10 augments CD40-induced expression of CD80, this cytokine inhibits the expression of CD80 that is induced by crosslinking CD120b. Further regulation of TNF-mediated CD80 expression may occur at the level of CD120b expression itself. We find that stimulation with exogenous IL-4 or CD40-cross-linking induces B cell expression of CD120b, but not CD120a. This study identifies an ancillary, TNF-mediated pathway, whereby activated T cells can induce B cells to express enhanced levels of the important costimulatory molecules, CD80 and CD54.

Mature dendritic cells (DCs), in addition to providing costimulation, can define the Th1, in contrast to the Th2, nature of a T-cell response through the production of cytokines and chemokines. Because calcium signaling alone causes rapid DC maturation of both normal and transformed myeloid cells, it was evaluated whether calcium-mobilized DCs polarize T cells toward a Th1 or a Th2 phenotype. After human monocytes were cultured for 24 hours in serum-free medium and granulocyte-macrophage colony-stimulating factor to produce immature DCs, additional overnight culture with either calcium ionophore (CI) or interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and soluble CD40L resulted in phenotypically mature DCs that produced interleukin-8 (IL-8) and displayed marked expression of CD80, CD86, CD40, CD54, CD83, DC-LAMP, and RelB. DCs matured by IFN-gamma, TNF-alpha, and soluble CD40L were additionally distinguished by undetectable CD4 expression, marked secretion of IL-12, IL-6, and MIP-1beta, and preferential ability to promote Th1/Tc1 characteristics during T-cell sensitization. In contrast, DCs matured by CI treatment were distinguished by CD4 expression, modest or absent levels of IL-12, IL-6, and MIP-1beta, and preferential ability to promote Th2/Tc2 characteristics. Calcium signaling selectively antagonized IL-12 production by mature DCs activated with IFN-gamma, TNF-alpha, and soluble CD40L. Although the activation of DCs by calcium signals is largely mediated through calcineurin phosphatase, the inhibition of IL-12 production by calcium signaling was independent of this enzyme. Naturally occurring calcium fluxes in immature DCs, therefore, negatively regulate Dc1 differentiation while promoting Dc2 characteristics and Th2/Tc2 polarization. Calcium-mobilized DCs may have clinical usefulness in treating disease states with excessive Th1/Tc1 activity, such as graft-versus-host disease or autoimmunity.

BACKGROUND

Allergen-specific conjunctival challenge (ASCC) is a safe and reproducible experimental model of allergic conjunctivitis and a useful tool in the evaluation of effectiveness and possible mechanisms of action of drugs commonly used in the treatment of allergic diseases.

OBJECTIVE

The protective effect of cetirizine on inflammatory changes after ASCC was assessed in 12 patients with rhinoconjunctivitis caused by Parietaria judaica in a double-blind study.

METHODS

After a screening ASCC was performed, patients were randomized into two treatment groups; each patient was given cetirizine (oral tablets) 10 mg twice daily, or matching placebo for 3 1/2 days in off-pollen season. Clinical evaluation (itching, hyperemia, lacrimation, and swelling of eyelids) and cytologic assessment (number of inflammatory cells in conjunctival scraping and evaluation of intercellular adhesion molecule-1 (ICAM-1)/CD54 expression on epithelial cells) were performed at baseline, 30 minutes (i.e., early-phase reaction [EPR]), 6 hours, and 24 hours (i.e., late-phase reaction [LPR]) after ASCC, before and after treatment.

RESULTS

The EPR clinical events and the EPR total number of inflammatory cells were significantly reduced by cetirizine compared with placebo. The LPR clinical events and inflammatory cell recruitment were reduced by cetirizine in a similar manner. Both eosinophil and neutrophil numbers were decreased by active drug in EPR and LPR. Furthermore, ICAM-1/CD54 expression was significantly reduced by cetirizine in both the EPR and LPR compared with placebo.

CONCLUSIONS

This study shows that cetirizine has a protective effect on clinical and cellular EPR and LPR events (including ICAM-1/CD54 expression on epithelium) induced by ASCC.

To determine whether part of the anti-inflammatory effects of prostaglandin E2 (PGE2) was related to inhibition of T cell interactions with endothelial cells (EC), the effects of PGE2 and other cAMP-elevating agents on the transendothelial migration of human T cells was examined. Although PGE2 did not effect T cell binding to EC, concentration-dependent inhibition of the transendothelial migration of T cells through unstimulated or IL-1-activated EC was observed. PGE2 inhibited the function of both T cells and EC, with maximal inhibition observed when both T cells and EC were treated with PGE2. However, the inhibitory action of PGE2 could not be ascribed to an effect on the adhesion receptor pair, CD11a/CD18-CD54. The inhibitory effect of PGE2 seemed to relate to its capacity to elevate cellular cAMP levels, because 3-isobutyl-1-methylxanthine enhanced PGE2 activity and dibutyryl cAMP and forskolin also inhibited transendothelial migration. The inhibitory effect of PGE2 and the other cAMP-elevating agents on the function of T cells related in part to suppression of their intrinsic locomotory behavior as random migration in the absence of EC was blocked. In EC, PGE2 and the other cAMP-elevating agents increased the barrier function of EC as evidenced by a decrease in the diffusion of [3H]mannitol through the endothelium. These results indicate that part of the anti-inflammatory action of PGE2 relates to its capacity to suppress the transendothelial migration of T cells by cAMP-mediated alterations in the function of both T cells and EC.

Reduced concentrations of glutamine (GLN) in plasma and skeletal muscle, defective host defense systems, and a diminished expression of the HLA-DR antigen on monocytes are important diagnostic parameters for late post-injury sepsis. In this in vitro study, we investigated whether blood monocyte-derived macrophage antigen expression and function from healthy donors is influenced by GLN. Lowering the GLN concentration in culture medium from 2 mmol/L to 200 mumol/L reduced the expression of HLA-DR by 40% (P < .001) on monocyte-derived macrophages, and decreased tetanus toxoid-induced antigen presentation. In addition, low GLN levels downregulated the expression of intercellular adhesion molecule-1 (ICAM-1/CD54), Fc receptor for IgG (Fc gamma RI/CD64), and complement receptors type 3 (CR3; CD11b/CD18) and type 4 (CR4; CD11c/CD18). A correlation was found between the phagocytosis of IgG-sensitized ox erythrocytes or opsonized Escherichia coli and the decreased expression of Fc gamma RI and CR3. Monocyte expression of CD14, CD71, and Fc gamma RIII/CD16 and capacity to phagocytose latex beads were not affected by altering the level of GLN. Depletion of GLN was associated with a significant reduction in cellular adenosine triphosphate (ATP), which may have influenced cell surface marker expression and phagocytosis. It remains to be seen whether these in vitro findings are of clinical significance in the treatment of sepsis.