Abstract: T-cell activation is a critical event in the organization of effective cellular and humoral immune responses. Activated T cells are essential for provision of T-cell help, promoting the development of high-affinity antibody production and the generation of cytotoxic T-cell responses. Accordingly, defects in proteins required for T-cell activation give rise to significant infectious pathology and malignancies. However, the decision to allow T-cell activation also has potentially dangerous consequences for the host and must therefore also be tightly controlled. Defects in proteins involved in regulating activated T-cell behaviour therefore tend to lead to autoimmunity. Thus, the major challenge faced in regulating T-cell responses is how to maintain a sufficiently large immune repertoire capable of recognizing all possible foreign antigens, whilst at the same time maintaining T cells in an unresponsive state towards self-antigens. In recent years significant progress has been made in our understanding of the mechanisms of self-tolerance. In the thymus it is clear that large numbers of potentially ‘self-reactive’ T cells are eliminated during negative selection in a process termed central tolerance. However, paradoxically, the process of positive selection that permits the expansion of T cells with low avidity for self–major histocompatibility complex (MHC) interactions must also lead to a degree of self-reactivity which is presumably tolerable in peripheral T cells. The question is how such T cells (albeit weakly self-reactive) can be ensured to remain non-reactive amongst a different array of self-antigens in the periphery. In the last few years a number of proteins have been identified that may serve the function of ‘quality controlling’ peripheral T-cell activation. This review focuses on two proteins, CD28 and cytotoxic T lymphocyte antigen-4 (CTLA-4), and explores how their interactions with their natural ligands may regulate the outcome of T-cell receptor engagement amongst peripheral T cells.

Abstract: Interleukin-16 (IL-16) acts as a chemoattractant for CD4+ cells, as a modulator of T-cell activation, and plays a key role in asthma. This report describes the cytokine-inducing effects of IL-16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4+ T lymphocytes did not secrete cytokines in response to rhIL-16, CD14+ CD4+ monocytes and maturing macrophages secrete IL-1beta, IL-6, IL-15 and tumour necrosis factor-alpha (TNF-alpha) upon rhIL-16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr post-stimulation, with protein being secreted by 24 hr. Secretion of IL-1beta and IL-6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL-16. However, for IL-15 or TNF-alpha maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL-16. Purified monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL-16, except for monocytes where maximal secretion of IL-15 was, interestingly, observed with only 50 ng/ml rhIL-16. The use of higher concentrations of rhIL-16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL-16-induced cytokines are likely to be involved in the immune system's response to antigen, the data suggest that IL-16 may play a key role in initiating and/or sustaining an inflammatory response.

Abstract: Human T cells expressing CD161 and an invariant T-cell receptor (TCR) alpha-chain (Valpha24invt T cells) specifically recognize CD1d and appear to have immunoregulatory functions. However, the physiological target cells for this T-cell population, and whether alterations in CD1d expression contribute to the regulation of Valpha24invt T-cell responses, remain to be determined. A series of antibodies were generated to assess CD1d expression, structure and regulation on human lymphoid and myeloid cells. CD1d was expressed at high levels by human cortical thymocytes and immunoprecipitation analyses showed it to be a 48 000-MW glycosylated protein. However, after solubilization, the majority of the thymocyte CD1d protein, but not CD1d expressed by transfected cells, lost reactivity with monoclonal antibodies (mAbs) against native CD1d, indicating that it was alternatively processed. Moreover, thymocytes were not recognized by CD1d-reactive Valpha24invt T-cell clones. Medullary thymocytes and resting peripheral blood T cells were CD1d-, but low-level CD1d expression was induced on activated T cells. CD1d was expressed by B cells in peripheral blood and lymph node mantle zones, but germinal centres were CD1d-. Resting monocytes were CD1d+ but, in contrast to CD1a, b and c, their surface expression of CD1d was not up-regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) activation. These results demonstrate constitutive CD1d expression by human professional antigen-presenting cells and that post-translational processing of CD1d may contribute to regulation of the activity of CD1d-specific T cells.

Abstract: In the armoury of the immune system developed to combat the various micro-organisms that could invade the host, the neutrophil forms the first line of defence against rapidly dividing bacteria and fungi. However, as humans age they become more susceptible to infection with these microbes and this has been ascribed to a decline in immune status, termed immune senescence. Here we summarize the literature specifically concerning the attenuation of neutrophil function with age and the possible mechanisms underlying their reduced response to infectious agents.

Abstract: Salmonella bacteria are a major cause of food-borne infectious diarrhoea and there is great interest in understanding the pathogenesis of Salmonella infection and in vaccine development Potential vaccines include the aromatic mutants of S typhimurium Such non-lethal Aro mutants have also been useful for studying Salmonella infections in mouse models Studies of systemic infection, using these Aro mutants, in both normal and cytokine gene knockout mice, indicate that interferon-gamma (IFN-gamma) plays a key role in the resolution of Salmonella infection The present studies have investigated the outcome of oral infection in mice with attenuated Salmonella because this infection route mimics natural infection in humans In IFN-gamma gene knockout (IFN-gamma-/-) mice, intestinal immunity was impaired and oral challenge resulted in disseminated septicaemia 2 weeks later No dissemination of infection was seen in wild-type mice In wild-type mice, both CD4 and CD8 cell numbers increased in the gut following Salmonella challenge, together with increased expression of major histocompatibility complex (MHC) II and vascular cell adhesion molecule-1 (VCAM-1) No such changes were seen in IFNgamma-/- mice Following oral challenge, antilipopolysaccharide (LPS) and antiphosphoryl choline antibodies increased by more than 100-fold in both serum and faecal pellet extracts of IFNgamma-/- mice compared with wild-type mice Our data show that IFN-gamma production is essential for resolution of enteric Salmonella infection and that antibody has little effect on this process

Abstract: Ficolins are a group of multimeric proteins that contain collagen-like and fibrinogen-like (FBG) sequences. Three types of ficolins have been characterized: H-, L- and M-ficolins. Both H- and L-ficolins have demonstrated lectin activities. In the present study, the FBG domain of M-ficolin was expressed and shown to bind to N-acetyl-D-glucosamine. M-ficolin mRNA was expressed in monocytes but not in the more differentiated macrophages and dendritic cells. By flow cytometry, surface biotinylation and immunoprecipitation, we showed that M-ficolin was associated with the surface of promonocytic U937 cells. M-ficolin transiently expressed in COS-7 cells was also clearly detected on the cell surface by immunoprecipitation. By flow cytometry, M-ficolin was detected on peripheral blood monocytes but not on lymphocytes or granulocytes. Immobilized rabbit anti-M-ficolin F(ab')2 mediated U937 cell adhesion, and the antibody also inhibited phagocytosis of Escherichia coli K-12 by U937 cells. Therefore, M-ficolin might act as a phagocytic receptor or adaptor on circulating monocytes for micro-organism recognition and may potentially mediate monocyte adhesion.

Abstract: This work examines the correlation between serum levels of oestrogen, progesterone and dehydroepiandrosterone sulphate (DHEA-S) and the number of human peripheral blood cells actively secreting interleukin (IL)-2, IL-4, IL-6, IL-10, tumour necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) in vivo. Simultaneous assessment of serum hormone levels and cytokine-secreting cell activity throughout the menstrual cycle showed that the number of peripheral blood mononuclear cells (PBMC) able to secrete IL-4 in response to stimulation correlated significantly (P < 0.0001) with oestrogen levels and fluctuated with the menstrual cycle in pre-menopausal women. The activity of IFN-gamma-secreting cells, on the other hand, varied as a function of serum DHEA-S levels in pre-menopausal women (P < 0.0001). Similarly, the number of cells secreting IFN-gamma in men correlated with serum DHEA-S levels (P < 0.001). In contrast, post-menopausal women had fewer cells actively secreting cytokines and the activity of these cells did not correlate with sex hormone levels. These results suggest that sex hormones may modulate cytokine production in vivo and contribute to gender-related differences in normal and pathological immune responses.

Abstract: Reactive oxygen species (ROS) are known to modulate activities of a host of kinases, phosphatases and transcription factors. Rutin and chlorogenic acid (CGA) are the major polyphenolic antioxidants present in the small molecular fraction of smokeless tobacco leaf extracts, as ascertained by reverse-phase high-pressure liquid chromatography (HPLC) and mass spectrometry. Levels of intracellular ROS in resting versus antigen-immunoglobulin E (IgE)-challenged murine mast cells were measured at 510 nm by fluorescence-activated cell sorting (FACS) using carboxy-dichlorofluorescein (DCFH-DA). Enhanced ROS production was observed in IgE-sensitized mast cells following antigenic challenge. Rutin and CGA reduced ROS levels in antigen-IgE-activated mast cells. Concomitantly, they also profoundly inhibited histamine release by these activated mast cells. In contrast, rutin and CGA augmented the inducible cytokine messages, i.e. interleukin (IL)-10, IL-13, interferon-gamma (IFN-gamma), IL-6 and tumour necrosis factor-alpha (TNF-alpha) in IgE-sensitized mast cells following antigen challenge. This study indicates that tobacco polyphenolic antioxidants that quench intracellular ROS, differentially affect two effector functions of antigen-IgE-activated mast cells. This model system may be employed to determine the molecular target of polyphenols. The potential role of these polyphenolic antioxidants on IgE-mediated allergy in vivo depends on a balance of their differential effects on mast cell activation.

Abstract: Compared with young mice, old mice infected with influenza virus have significantly higher pulmonary viral titres, although these can be reduced significantly with dietary vitamin E supplementation. T helper 1 (Th1) cytokines, especially interferon-gamma (IFN-gamma), play an important role in defending against influenza infection. However, there is an age-associated loss of Th1 cytokine production. Prostaglandin E2 (PGE2) production, which increases with age, can modulate the T helper cell function by suppressing Th1 cytokine production. To investigate the mechanism of vitamin E supplementation on reduction of influenza severity in old mice, we studied the cytokine production by splenocytes, and PGE2 production by macrophages (Mphi), in young and old C57BL mice fed semipurified diets containing 30 (control) or 500 parts per million (ppm) (supplemented) vitamin E for 8 weeks, and then infected with influenza A/PC/1/73 (H3N2). Old mice fed the control diet had significantly higher viral titres than young mice; old mice fed the vitamin E-supplemented diet had significantly lower pulmonary viral titres than those fed the control diet (P = 0.02 and 0.001 for overall age and diet effect, respectively). Following influenza infection, interleukin (IL)-2 and IFN-gamma production was significantly lower in old mice than in young mice. Vitamin E supplementation increased production of IL-2 and IFN-gamma in old mice; higher IFN-gamma production was associated with lower pulmonary viral titre. Old mice fed the control diet showed significantly higher lipopolysaccharide (LPS)-stimulated Mphi PGE2 production than old mice fed the vitamin E diet or young mice fed either diet. There was no significant age difference in IL-6, IL-1beta, or tumour necrosis factor-alpha (TNF-alpha) production by splenocytes. Young mice fed the vitamin E-supplemented diet had significantly lower IL-1beta (day 7) and TNF-alpha production (day 5) compared with those fed the control diet. Old mice fed the vitamin E-supplemented diet had significantly lower TNF-alpha production (day 2) than those fed the control diet. Our results indicate that the vitamin E-induced decrease in influenza viral titre is mediated through enhancement of Th1 cytokines, which may be the result of reduced PGE2 production caused by vitamin E.

Abstract: Following injection challenge of rainbow trout with the Gram-positive pathogen Renibacterium salmoninarum, serum nitrate levels increased indicative of NO production. The timing and amount of nitrate produced varied with the virulence of the bacterial strain used, with the highest levels seen in fish challenged with the most virulent (autoaggregating) strain. Immunization with a killed R. salmoninarum preparation in Freund's incomplete adjuvant significantly increased nitrate levels after challenge. Inducible nitric oxide synthase (iNOS) transcript expression was detectable in rainbow trout tissues after injection challenge with R. salmoninarum, and its induction in the gills was both quick (between 3 and 6 hr) and relatively prolonged (lasting several days). iNOS expression in the kidney was also seen at a later stage (24 hr) but appeared to switch off relatively rapidly. Bath challenge with R. salmoninarum also induced iNOS expression in gill, and a variable expression in the gut and kidney also occurred. These results highlight the importance of the gills, not only as a point of entry of pathogens but also as a tissue capable of mounting an immune response.

Abstract: Summary Activated dendritic cells are critically important in the priming of T-cell responses. In this report we show that the infection of a conditionally immortalized dendritic cell line (tsDC) with Mycobacterium tuberculosis resulted in the up-regulation of B7-1 and B7-2 co-stimulatory molecules and the induction of several inflammatory cytokines, including tumour necrosis factor-α and interleukin-6, -1β and -12. In addition, we show that these activated dendritic cells were capable of eliciting antigen-specific T-cell responses and potent anti-mycobacterial protective immunity in a murine model of experimental tuberculosis infection.

Abstract: The role of cytokines in the pathogenesis and immunity of Marek's disease (MD), a herpesvirus-induced T-cell lymphoma in chickens, is poorly understood. Two different experiments were used to examine the potential role of particular cytokines in the pathogenesis and immune responses of MD. First, chicken embryo fibroblasts (CEF) were stimulated with lipopolysaccharide (LPS) and/or recombinant chicken interferon-gamma (rChIFN-gamma) and used to develop techniques for examining transcription of IFN-alpha, IFN-gamma, inducible nitric oxide synthase (iNOS), interleukin (IL)-1beta, IL-2, IL-6 and IL-8 by reverse transcription-polymerase chain reaction (RT-PCR). Addition of LPS and/or rChIFN-gamma resulted in the up-regulation of mRNA for iNOS, IL-1beta and IL-6, while IFN-gamma was up-regulated by LPS alone. IL-2 was down-regulated by the treatments. Second, to determine the effects of Marek's disease herpesvirus (MDV) infection on cytokine transcription in vivo, chickens were infected with MDV at 21 days of age and examined at 7 days post-infection (p.i.) (exp. 1) or were infected with MDV at 1 day of age and examined from 3 to 15 days p.i. (exp. 2). In MDV-infected chickens, IFN-gamma transcription was up-regulated as early as 3 days p.i. until the termination of the experiment at 15 days p.i., while iNOS and IL-1beta were up-regulated between 6 and 15 days p.i. Infection of 1-day-old chicks increased levels of mRNA for IFN-gamma and iNOS between 16- and 64-fold at 9 days p.i. These results suggest that IFN-gamma and iNOS may play an important role in the pathogenesis of MD.

Abstract: The delivery of antigens to mucosal-associated lymphoid tissues in paediatric and immunocompromised populations by safe, non-invasive vectors, such as commensal lactobacilli, represents a crucial improvement to prevailing vaccination options. In this report, we describe the oral and nasal immunization of mice with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 000-molecular weight (MW) fragment C of tetanus toxin (TTFC) is expressed either as an intracellular or a surface-exposed protein. Our data indicate that L. plantarum is more effective in this respect than L. casei and that, under the experimental conditions investigated, delivery of TTFC expressed as an intracellular antigen is more effective than cell-surface expression. Immunization of mice with live recombinant lactobacilli induced significant levels of circulating TTFC-specific immunoglobulin G (IgG) following nasal or oral delivery of vaccine strains. In addition, following nasal delivery, secretory immunoglobulin A (sIgA) was induced in bronchoalveolar lavage fluids, as were antigen-specific antibody-secreting cells and antigen-specific T-cell activation in draining lymph nodes, substantiating their potential for safe mucosal delivery of paediatric vaccines.

Abstract: Human Valpha24 + NKT cells, a subpopulation of natural killer cell receptor (NKR-P1A) expressing T cells with an invariant T-cell receptor (TCR; Valpha24JalphaQ) are stimulated by the glycolipid, alpha-galactosylceramide (KRN7000), in a CD1d-dependent, TCR-mediated fashion. Little is known about Valpha24 + NKT-cell function. The murine counterpart, Valpha14 + NKT cells, appear to have an important role in controlling malignancy. There are no human data examining the role of Valpha24 + NKT cells in controlling human malignancy. We report that Valpha24 + NKT cells have perforin-mediated cytotoxicity against haemopoietic malignancies. Valpha24 TCR, CD1d and alpha-galactosylceramide may all play a role in cytotoxicity but are not absolute requirements. The greatest cytotoxicity was observed against the U937 tumour cell line (95 +/- 5% lysis). THP-1, Molt4, C1R cells and allogeneic mismatched dendritic cells were also sensitive to Valpha24 + NKT cytotoxicity but neither the NK target, K562, nor lymphokine-activated killer-sensitive Daudi cells, were sensitive. These results indicate a killing pattern distinct from conventional major histocompatibility complex-restricted T cells, NK cells and other cytotoxic lymphoid cells previously described. We conclude that human Valpha24 + NKT cells have cytotoxic anti-tumour activity against haemopoietic malignancies through effector mechanisms distinct from conventional T cells and NK cells and that their specific stimulator KRN7000 may have therapeutic potential.

Abstract: SUMMARY Multinucleated giant cells (MGC) are a common feature of granulomas. The mechanism of their formation has been studied extensively, but their function has not been completely characterized. A new method for the in vivo production of MGC was developed involving subcutaneous injection of microscopic nitrocellulose particles with adsorbed mycobacterial antigens into the footpads of sensitized BALB/c mice (immune [I]-MGC), or by nitrocellulose administration to non-sensitized mice (foreign body [FB]-MGC). The development of granulomas with a highly enriched MGC population was observed 2 weeks after the nitrocellulose injection. MGC were larger with a greater number of nuclei in I-MGC than in FB-MGC. From days 7‐28 after nitrocellulose administration, the production of interleukin-1a (IL-1a) and tumour necrosis factor-a (TNF-a) was demonstrated in both MGC types by in situ reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemistry. After 2 months, the MGC had ceased production of IL-1a and TNF-a, but the expression of transforming growth factor-b (TGF-b) was very high, occurring together with extensive fibrosis. These results suggest that MGC are an active source of inflammatory cytokines, which can contribute to the initiation, maintenance and down-regulation of granulomatous inflammation induced by immunological and inert substances.

Abstract: Dendritic cells (DC) have recently been shown to play an important role in B-cell function. We have previously shown that DC can capture and retain unprocessed antigen in vitro and in vivo, and can transfer this antigen to naive B cells to initiate antigen-specific antibody responses. We also demonstrated that DC were providing B cells with isotype-switch signals independent of T cells but that T-cell help was essential for antibody production. In this study, using B cells and DC from wild type (WT) and CD40 knockout (CD40KO) mice we show that DC initiate proliferation of B cells independently of CD40, because WT or CD40KO DC could induce proliferation of WT or CD40KO B cells, but proliferation was greater in the absence of CD40. DC also provide B cells with survival signals as WT DC improved viability of B cells after a 5-day culture but survival was reduced in the absence of CD40 expression.

Abstract: Interleukin-10 (IL-10) down-regulates T helper type 1 cell and macrophage functions. As IL-10 is induced along with tumour necrosis factor (TNF) and IL-12 in mycobacterial infection, we asked whether endogenous IL-10 plays a role in the antimycobacterial response. We demonstrate here that IL-10-deficient mice eliminate Mycobacterium bovis Calmette-Guerin bacillus faster than wild-type mice. Granulomas are significantly larger, containing more CD-11b- and CD11c-positive antigen-presenting cells and T cells, and the expression of major histocompatibility complex class II and intracellular adhesion molecule-1 is increased. Macrophages in granulomas of IL-10-deficient mice express high levels of TNF, acid phosphatase and inducible nitric oxide synthase (iNOS). Finally, an increased cutaneous delayed-type hypersensitivity reaction to mycobacterial proteins is further evidence of an augmented cell-mediated immune response. In conclusion, the cell-mediated immunity is enhanced in the absence of IL-10, resulting in a robust granuloma response, which accelerates the clearance of mycobacteria. Therefore, endogenous IL-10 attenuates mycobacterial immunity.

Abstract: Immune responses can be classified, according to the predominant cytokines involved, into type 1 (featuring interferon-gamma, IFN-gamma) and type 2 (featuring interleukin-4, IL-4); imbalance between type 1 and type 2 cytokine compartments has been implicated in many human diseases. Levamisole is a drug with an unknown mode of action that has been used to boost immunity in infectious diseases including leprosy, and in some cancers. To test the hypothesis that levamisole acts by inducing a shift to a type 1 immune response, we used Brown Norway (BN) rats, which are markedly biased to type 2 responses. BN rats treated with levamisole showed a dose-dependent rise in serum IFN-gamma and fall in serum immunoglobulin E (IgE) level. Detailed analysis of cytokine gene expression showed upregulation of IFN-gamma and downregulation of IL-4 messenger RNA. This coincided with marked upregulation of IL-18, a recently characterized cytokine with potent activity in stimulating IFN-gamma production. IL-12 was not induced. Further, the type 2 response induced in BN rats by mercuric chloride was markedly attenuated when rats were pretreated with levamisole: there was a 2-log reduction in maximum serum IgE level and marked attenuation of IL-4 gene upregulation. These data indicate that levamisole acts by resetting the immune balance towards a type 1 response via induction of IL-18. Our findings provide a direction for development of more specific immunomodulating therapy.

Abstract: The Brambell receptor (FcRB) mediates functions of both immunoglobulin G (IgG) transport, transmitting immunity from mother to young, and IgG protection, making IgG the longest surviving of all plasma proteins. Reflecting its role as transport receptor (termed FcRn, for neonatal rat intestine, the tissue from which it was first cloned), FcRB is expressed antenatally in the rabbit, mouse and rat fetal yolk sac and in human placental syncytiotrophoblasts, and neonatally in the intestinal epithelium of mice and rats. Reflecting its role as protection receptor (FcRp), FcRB is expressed in the vascular endothelium throughout life, where it protects IgG from the on-going catabolic activities of this tissue. FcRB detected in hepatocytes was hypothesized to mediate transport of IgG from serum to bile, thus potentially extending the transport expression (FcRn) of this receptor beyond the perinatal period. Our results show serum-to-bile transport of IgG to be unaffected in mice functionally deleted for FcRB. Accordingly, the hypothesis is rejected that FcRB functions as transport receptor (FcRn) in liver. The default conclusion is that FcRB in hepatocytes functions as FcRp, serving to protect IgG from catabolism in hepatocytes that accompanies the endocytic activity of these cells. We conclude that there remains to date no evidence of an FcRn-like transport function of the Brambell receptor beyond the perinatal period, after which the FcRp function of the receptor predominates, paralleling the endocytic activities of the associated tissues.

Abstract: The mucosal immunogenicity of a number of plant lectins with different sugar specificities was investigated in mice. Following intranasal (i.n.) or oral administration, the systemic and mucosal antibody responses elicited were compared with those induced by a potent mucosal immunogen (cholera toxin; CT) and a poorly immunogenic protein (ovalbumin; OVA). After three oral or i.n. doses of CT, high levels of specific serum antibodies were measured and specific IgA was detected in the serum, saliva, vaginal wash, nasal wash and gut wash of mice. Immunization with OVA elicited low titres of serum IgG but specific IgA was not detected in mucosal secretions. Both oral and i.n. delivery of all five plant lectins investigated ?Viscum album (mistletoe lectin 1; ML-1), Lycospersicum esculentum (tomato lectin; LEA), Phaseolus vulgaris (PHA), Triticum vulgaris (wheat germ agglutinin (WGA), Ulex europaeus I (UEA-1) stimulated the production of specific serum IgG and IgA antibody after three i. n. or oral doses. Immunization with ML-1 induced high titres of serum IgG and IgA in addition to specific IgA in mucosal secretions. The response to orally delivered ML-1 was comparable to that induced by CT, although a 10-fold higher dose was administered. Immunization with LEA also induced high titres of serum IgG, particularly after i. n. delivery. Low specific IgA titres were also detected to LEA in mucosal secretions. Responses to PHA, WGA and UEA-1 were measured at a relatively low level in the serum, and little or no specific mucosal IgA was detected.

Abstract: When immature human myeloid dendritic cells were differentiated in vitro in the presence of aspirin, they were unable to stimulate T-cell proliferation. Aspirin and its major metabolite salicylate changed the surface marker phenotype of dendritic cells. The drugs particularly suppressed the levels of CD83 and the secreted p40 unit of interleukin-12 (IL-12), both markers of mature dendritic cells; 50% inhibitory concentration (IC50) values were 2.5 mM, a concentration more than 100 times greater than the concentration at mid-point inhibition (ID50) value for inhibition of prostaglandin synthesis. Concomitantly, the levels of CD14, a marker of monocytes/macrophages, increased above the levels found in immature dendritic cells. Cyclooxygenase inhibitors ketoprofen, indomethacin and NS-398 had no effect at concentrations more than a thousand-fold higher than their IC50 values. The effects were independent of the presence of prostaglandin E2 in the medium. Salicylates suppressed activation of the nuclear transcription factor kappaB, which regulates dendritic cell differentiation, but their effects on mature dendritic cells were negligible. Hence, aspirin inhibits dendritic cell function by inhibiting their terminal differentiation at concentrations achieved in the blood of patients chronically treated with high-dose aspirin.

Abstract: We previously reported that CA074, a specific inhibitor of cathepsin B, modulates specific immune responses from the T helper 2 (Th2) type to Th1 type in BALB/c mice infected with Leishmania major. In the present study, we found that a similar type of immune deviation was also induced in mice immunized with ovalbumin (OVA). However, treatment of mice with pepstatin A, a specific cathepsin D inhibitor, suppressed the OVA-specific proliferation of lymphocytes and blocked the development of both Th1 and Th2 cellular responses. These inhibitors did not appear to have any direct influence in vitro on functions of naive lymphocytes. OVA antigen (47 000 MW) was digested mainly into 40 000 MW protein in vitro by lysosomal proteases from naive BALB/c mice, and its digestion was markedly inhibited by the addition of CA074, but not by addition of pepstatin A, during incubation. However, pepstatin A strongly suppressed the degradation of the major histocompatibility complex class II-associated invariant chain (Ii) molecule in vivo and in vitro. Thus, cathepsin B appears to process antigens directed to preferential activation of Th2 cells, while cathepsin D may be responsible for the degradation of Ii, the processing of which is essential in initiating the antigen-specific activation of Th1 and Th2 CD4+ T cells. These lysosomal proteases may have different functions in regulating immune responses.

Abstract: Human monocyte-derived dendritic cells (MoDCs) obtained from peripheral blood monocytes (PBMC) cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) can be activated in vitro by a variety of simple chemicals such as haptens and several metals. Recently, it has been demonstrated that transforming growth factor-beta1 (TGF-beta1) can induce further differentiation of MoDCs to the cells that share some characteristics with epidermal Langerhans cells, i.e. they contain Birbeck granules and express E-cadherin. In this study, using such TGF-beta1-treated dendritic cells (TGF-beta1+ DCs), we examined the in vitro effects of representative haptens, i.e. NiCl2 and dinitrochlorobenzene (DNCB), on their phenotypic and functional characteristics, comparing with those reported in vivo in epidermal Langerhans cells during the sensitization phase of a contact sensitivity reaction. Treatment of TGF-beta1+ DCs with NiCl2 increased their expression of the molecules related to antigen presentation such as CD86, major histocompatibility complex class I and class II, and CD83, although weakly, in addition to that of those essential for their migration to the regional lymph nodes, such as CD49e, CD44 and its variant 6, while it down-regulated the expression of the molecules required for homing to the skin and staying in the epidermis, such as cutaneous leucocyte antigen (CLA) and E-cadherin. It also increased the production of tumour necrosis factor-alpha, but not that of IL-1beta or IL-12. DNCB also increased their CD86 expression and down-regulated E-cadherin and CLA, but did not affect other phenotypic changes that were observed in TGF-beta1+ DCs treated with NiCl2. TGF-beta1+ DCs treated with either NiCl2 or DNCB increased their allogeneic T-cell stimulatory function. In addition, reverse transcribed polymerase chain reaction revealed augmented expression of chemokine receptor 7 mRNA by TGF-beta1+ DCs when treated with either NiCl2 or DNCB. Moreover, consistent with this data, TGF-beta1+ DCs treated with these chemicals chemotactically responded to macrophage inflammatory protein-3beta. These data suggest the possibility that TGF-beta1+ DCs present a good in vitro model to study the biology of epidermal Langerhans cells.

Abstract: SUMMARY Chemokine production at the blood‐retina barrier probably plays a critical role in determining the influx of tissue-damaging cells from the circulation into the retina during inflammation. The blood‐ retina barrier comprises the retinal microvascular endothelium and the retinal pigment epithelium. Chemokine expression and production by human retinal microvascular endothelial cells (REC) have never been reported previously, so we examined the in vitro expression and production of monocyte chemoattractant protein-1 (MCP-1), regulated on activation of normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1a, MIP-1b, interleukin (IL)-8, epithelial cell-derived neutrophil activating protein-78 (ENA-78) and growth related oncogene a (GROa) in these cells, both unstimulated and stimulated by cytokines likely to be present during the evolution of an inflammatory response. We compared this to expression and production of these chemokines in vitro in human retinal pigment epithelial cells (RPE). MCP-1 was expressed and produced constitutively by REC but all the chemokines were produced in greater amounts upon stimulation with the proinflammatory cytokines IL-1b and tumour necrosis factor-a (TNF-a). MCP-1 and IL-8 were produced at much higher levels than the other chemokines tested. MIP-1a and MIP-1b were present only at low levels, even after stimulation with IL-1b and TNF-a. Cytokines with greater anti-inflammatory activity, such as IL-4, IL-10, IL-13, transforming growth factor-b (TGF-b) and IL-6, had little effect on chemokine production either by REC alone or after stimulation with IL-1b and TNF-a. RPE, although a very different cell type, showed a similar pattern of expression and production of chemokines, indicating the site-specific nature of chemokine production. Chemokine production by REC and RPE is probably significant in selective leucocyte recruitment during the development of inflammation in the retina.

Abstract: Homologous complement activation is restricted on cells by the complement regulators, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. These proteins act in concert with other membrane structures to protect cells from homologous complement attack. In contrast, cells are usually sensitive to heterologous complement attack. It has been suggested that species-specific restriction of complement activation can be attributed to the inability of regulators to inhibit across species. We have investigated the capacities of human, rat and mouse analogues of DAF to regulate homologous and heterologous complement. Cells transfected with cDNA encoding these analogues were protected from heterologous complement attack. C3b-deposition experiments indicated that whilst cells were best protected by DAF from the same species, all three analogues inhibited human, rat and mouse complement. Comparable results were obtained in haemolysis assays using soluble, recombinant forms of the proteins. Inhibition of the classical pathway (CP) was best achieved with homologous DAF, although human DAF also inhibited rat complement, rat DAF also inhibited human complement and mouse DAF inhibited complement from all species. Human DAF was the best inhibitor of alternative pathway (AP)-mediated attack, inhibiting complement from all species. Mouse DAF inhibited mouse and rat AP, whilst rat DAF inhibited only rat AP. These data indicate that human and rodent analogues of DAF are not species restricted and highlights interesting differences in the capacity to regulate AP and CP. This has implications in broader fields of research, such as xenotransplantation, where cross-species regulation of complement is of paramount importance.

Abstract: Chicken antibodies are increasingly being used as diagnostic and therapeutic tools. As only the genomic organization of the micro encoding gene was previously known, we analysed the chicken immunoglobulin Y (IgY) and IgA (upsilon and alpha chain) immunoglobulin heavy-chain constant region (IGHC) genes and the organization of the chicken IGHC locus. The alpha gene is encoded by four separate exons, whereas, surprisingly, there is no intervening DNA sequence between the CH1 and CH2 domains of the IgY heavy chain, which is thus encoded by three exons separated by two introns. DNA sequence analysis shows that the exon boundaries of the chicken IGHC genes are not consistent with published domain borders. Furthermore, differences in DNA sequence confirm the existence of IgY, IgA and IgM allotypes in chickens. Finally, our results show that the IGHC genes of chicken (IgY, IgA and IgM) are all colocated on chromosome E18C15W15, where the alpha gene is located upstream of the upsilon gene in an inverted transcriptional orientation. The distances between the mu and alpha genes and the alpha and upsilon genes are about 18 and 15 kilobases, respectively, and thus, the size of the whole chicken IGHC locus is approximately 67 kilobases.

Abstract: Previously we showed that calcitonin gene-related peptide (CGRP), a neuropeptide, inhibited lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) production and increased interleukin (IL)-6 release at low concentrations via activation of the cAMP pathway in mouse peritoneal macrophages (Mphi). In this study we examined whether CGRP could modulate IL-12 release from mouse peritoneal Mphi, and if so, what signal transduction pathway was involved. Mphi were obtained from the peritoneal exudate of male BALB/c mice. The cells were plated on culture dishes at a density of 5 x 105 cells per well and allowed to adhere for 2 hr. After incubation for 24 hr, the Mphi were cultured with 0.1 microg/ml of LPS, alone or together with CGRP (1-1000 nM) for 24 hr. The amount of IL-12 in the cell medium was measured by enzyme-linked immunosorbent assay (ELISA). The results showed that CGRP attenuated LPS-induced IL-12 release in a concentration-dependent manner. Production of IL-12 was decreased from 95.9+/-4.6 to 73.4+/-5.7 pg/ml by 100 nM CGRP. The two cAMP phosphodiesterase (PDE) inhibitors, 3-isobutyl-1-methyl-xanthine (IBMX) and rolipram, significantly potentiated the CGRP response, and the level of IL-12 was further decreased by 28% and 47%, respectively. However, CGRP had no effect on IL-12 production from unstimulated Mphi. The LPS-induced IL-12 release from Mphi could also be reduced by forskolin, an activator of adenylate cyclase, and 8-Br-cAMP, an analogue of cAMP. Using the reverse transcription-polymerase chain reaction (RT-PCR), we found that CGRP also decreased the LPS-induced IL-12 p40 mRNA levels. Furthermore, pretreatment with H89 (0.1 microM or 1 microM), an inhibitor of cAMP-dependent protein kinase, diminished CGRP effects, IL-12 production and gene expression. These data suggest that LPS-induced IL-12 release and gene expression were attenuated by CGRP via an activated cAMP-protein kinase A (PKA) pathway in mouse peritoneal Mphi.

Abstract: Dendritic cells (DC) are important initiators of an immune response so understanding the factors controlling antigen acquisition and presentation has important consequences for the use of these cells in vaccines and other forms of immunotherapy. We investigated the factors that influence phagocytosis by immature bone marrow-derived DC (BMDC) and the effect of interleukin-10 (IL-10) on this process. Two sizes of fluorescent particles and recombinant bacillus Calmette-Guerin expressing the green fluorescent protein (rBCG) were used as particulate antigens. The percentage of cells taking up the antigen was found to be dependent on the size and dose of the particles, and the length of exposure to them. BMDC exposed to IL-10 at various concentrations for different periods exhibited no distinguishable change in antigen uptake. However, if BMDC treated with IL-10 and rBCG were then exposed to a second dose of particulate antigen, uptake was increased compared with those BMDC not treated with IL-10. The expression of major histocompatibility complex class II, CD80, CD86 and CD11c by BMDC after phagocytosing rBCG or inert beads, was inhibited when the BMDC were pretreated with IL-10. In contrast, the expression of CD25 was increased. BMDC that had taken up BCG or purified protein derivative (PPD) were able to stimulate primed T-cell proliferation but this was severely inhibited if the BMDC were cultured with IL-10 before exposure to the antigen. This work suggests that although IL-10 does not affect the phagocytic capacity of BMDC, it does inhibit maturation of the cells and consequently, T-cell activation.

Abstract: We have previously presented evidence of a highly organized and compartmentalized structure of the small intestinal lamina propria of the pig. In this work, we have identified at least two major populations of cells in this site expressing high levels of major histocompatibility complex (MHC) class II antigens. One is CD45 positive and is a potent initiator of a primary immune response, this is a function usually associated with dendritic cells. These cells have characteristic dendritic morphology, but show evidence of phagocytosis as well as other phenotypic markers of immature dendritic cells. Some cells show evidence of ongoing immune maturation. We have also isolated CD45 negative endothelial cells bearing significant amounts of MHC class II, which do not trigger a mixed lymphocyte reaction. These findings have implications for the functional role of healthy gut lamina propria and clearly implicate this site as capable of differential antigen presentation by a heterogeneous population of antigen-presenting cells.