concanavalin-a and Granuloma

Nephronectin (Npnt) is an extracellular matrix protein known to play a critical role in kidney development; however, its physiological role in the liver remains elusive. Here we show that Npnt expression is upregulated in mouse models of both acute and chronic hepatitis induced by Concanavalin A (Con A) and 3,5-diethocarbonyl-1,4-dihydrocollidine (DDC), respectively. In both models, Npnt was localized in inflammatory foci and was mainly secreted from mesenchymal cells and in part by cholangiocytes. Interestingly, ectopic expression of Npnt in hepatocytes induced the development of granuloma-like cell clusters mainly composed of CD4(+) T cells or NKT cells but did not induce apparent hepatitis. Furthermore, we found that Npnt exacerbated the Con A-induced acute hepatitis. These results indicate that Npnt plays an important role in the initiation of hepatitis by recruiting CD4(+) T cells or NKT cells into the foci of inflammation. In addition, we reveal that Npnt expression is also upregulated in human hepatitis. Therefore, Npnt may be a potential therapeutic target for acute and chronic hepatitis.

Topics: Acute Disease; Animals; CD4-Positive T-Lymphocytes; Cell Movement; Concanavalin A; Disease Models, Animal; Disease Progression; Extracellular Matrix Proteins; Gene Expression Regulation; Granuloma; Hepatitis; Hepatitis, Chronic; Liver; Male; Mice; Mice, Inbred C57BL; Up-Regulation

There have been several attempts to make granuloma model to clarify the mechanism of granulomatous diseases like sarcoidosis. However, a unique in vitro model that generates multinucleated giant cell (MGC) through epithelioid cells resembled to human granuloma, has not yet been clearly established. In this study, the generation of granuloma model that forms MGC via epithelioid cells from the mouse macrophage cell line was investigated. A RAW 246.7 mouse macrophage cell line was cultured with lipopolysaccharide (LPS) and concanavalin A (Con A) in various concentrations either alone or both. We found that separate treatment of LPS and Con A induced around 35 and 20% MGC respectively whereas cotreatment of these chemicals drastically accelerated granuloma formation rate and it was around 80%. The highest fusion index (MGC formation rate) was observed at days 7. A gradual increase of tumor necrosis factor alpha (TNF-alpha) production in the culture supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA). And the neutralization of the elevated level of TNF-alpha production by its monoclonal antibody leads to significant decrease of MGC formation. Interestingly, we found that the RAW cells were changed into spindle cells, which morphologically resembled to epithelioid cells and eventually MGC was formed from these spindle cells. Our in vitro granuloma model appeared to be similar with in vivo epithelioid cell granulomas like sarcoidosis. Thus, our model would be useful as in vitro epithelioid granuloma model for analyzing the mechanisms and screening the effective drugs of granulomatous diseases in future.

Topics: Animals; Cell Line; Concanavalin A; Culture Techniques; Giant Cells; Granuloma; Lipopolysaccharides; Macrophages; Mice; Mitogens; Tumor Necrosis Factor-alpha

The glucocorticoid ciclesonide is the 2'R-epimer of 2'-cyclohexyl-11beta-hydroxy-21-isobutyryloxy-16bH-dioxolo[5',4':16,17]pregna-1,4-diene-3,20-dione. The active metabolite desisobutyryl-ciclesonide (des-CIC) is derived from ciclesonide by esterase cleavage of isobutyrate at the C21 position. The relative binding affinities at the rat glucocorticoid receptor were dexamethasone, 100; ciclesonide, 12; des-CIC, 1212; and budesonide, 905. Des-CIC potently inhibited the activation of murine and human lymphocytes in a series of different in vitro systems. With the exception of concanavalin A-stimulated rat spleen cells, des-CIC was more potent than the parent compound. Des-CIC compared well with budesonide in all in vitro systems. Furthermore, the respective 2'S-epimers were always significantly less potent than the 2'R-epimers. In vivo, ciclesonide (intratracheal administration), des-CIC, and budesonide inhibited antigen-induced accumulation of eosinophils, protein, and tumor necrosis factor-alpha into the bronchoalveolar lavage fluid of ovalbumin-sensitized and -challenged Brown Norway rats with an ED(50) value ranging from 0.4 to 1.3 mg/kg, indicating similar potency, which suggests in vivo activation of the parent compound. Ciclesonide and budesonide inhibited the bradykinin-induced protein leakage into the rat trachea. In the rat cotton pellet model, ciclesonide inhibited granuloma formation (ED(50):= of 2 microg/pellet), whereas budesonide and des-CIC were 15- and 20-fold less active; thymus involution was induced with an ED(50) of 303, 279, and 154 microg/pellet, respectively. When applied orally to rats for 28 days, ciclesonide showed low potency in reducing weight of thymus and adrenals, suggesting low oral bioavailability. The in vivo data on ciclesonide highlight its effective local action and a reduced potential for side effects.

Topics: Administration, Oral; Administration, Topical; Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Binding Sites; Bradykinin; CD3 Complex; CD4-Positive T-Lymphocytes; Cell Division; Concanavalin A; Cytokines; Glucocorticoids; Granuloma; Humans; Interleukin-4; Interleukin-5; Leukocytes, Mononuclear; Lung; Male; Neoplasm Transplantation; Neoplasms, Experimental; Pregnenediones; Rats; Rats, Sprague-Dawley; Receptors, Glucocorticoid; Spleen; Trachea

Reinfection with Schistosoma mansoni following chemotherapy often results in an ameliorated granulomatous reaction and hence a mild disease. This study examined some of the immunological mechanisms that could be associated with this residual protection. BALB/c mice were infected with either a single dose (group A) of 100 S. mansoni cercariae or with 10 doses of 10 cercariae each (group B) given at 3-day intervals. The mice were treated with praziquantel 8 weeks postinfection and, 2 weeks later, together with another group of naive mice (group C), they were infected with a single dose of 100 cercariae each. All the animals were killed 8 weeks later and schistosome egg antigen (SEA)- and soluble adult worm antigen preparation (SWAP)-induced cytokine recall responses in splenocytes, as well as serum immunoglobulin levels, were quantified and hepatic granuloma sizes measured. Group A animals had higher levels of SEA-induced interferon-gamma (IFN-gamma) but lower levels of interleukin (IL)-5 than groups B and C (P < 0.01). Group B animals had low SEA-induced IFN-gamma levels and elevated IL-5 levels, although these were lower than group C. SEA-induced IL-10 was low in both groups A and B as compared to group C (P < 0.01). SWAP was less effective as an inducer of splenocyte cytokine production than SEA but both SWAP-induced IFN-gamma and IL-5 were detected in groups A and C. SEA- and SWAP-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) titres were not significantly different between the three groups. Granuloma diameters were larger in group C (mean 297 +/- 51.3 microm) as compared to groups A (174 +/- 49 microm, P < 0.01) and B (247.5 +/- 44 microm, P < 0.05). Taken together, these results demonstrate that granuloma size is reduced during a reinfection exposure compared with a primary infection. This reduction is associated with a T helper 1 response in mice exposed to a single large dose of cercariae in the primary infection and with a predominantly T helper 2 response in those infected with multiple small doses.

Topics: Animals; Antibodies, Helminth; Antigens, Helminth; Cells, Cultured; Concanavalin A; Culture Media; Cytokines; Female; Granuloma; Liver Diseases, Parasitic; Mice; Mice, Inbred BALB C; Praziquantel; Recurrence; Schistosoma mansoni; Schistosomiasis mansoni; Spleen; Th1 Cells; Th2 Cells

Thirty-nine patients with fibromyalgia syndrome (FMS) according to American College of Rheumatology criteria were studied for cell-mediated sensitivity to environmental chemicals. Lymphocytes were tested by standard [(3)H]thymidine incorporation in vitro for T cell memory to 11 chemical substances. Concanavalin A (Con A) was used to demonstrate T cell proliferation. Controls were 25 contemporaneous healthy adults and 252 other concurrent standard controls without any aspect of FMS. Significantly higher (P < 0.01) stimulation indexes (SI) were found in FMS for aluminum, lead, and platinum; borderline higher (0.05 > P > 0.02) SI were found for cadmium and silicon. FMS patients showed sporadic responses to the specific substances tested, with no high-frequency result (>50%) and no obvious pattern. Mitogenic responses to Con A indicated some suppression of T cell functionality in FMS. Possible links between mitogenicity and immunogenic T cell proliferation, certain electrochemical specifics of granuloma formation, maintenance of connective tissue, and the fundamental nature of FMS are considered.

Topics: Adult; Aged; Antigens; Case-Control Studies; Concanavalin A; Female; Fibromyalgia; Granuloma; Humans; In Vitro Techniques; Lymphocyte Activation; Male; Metals; Middle Aged; Siloxanes; T-Lymphocytes

Synchronized liver granulomas were induced by injecting Sepharose beads to which SEA soluble egg antigen (SEA) or the concanavalin A binding fraction of SEA had been coupled into a mesenteric vein in naive, single-sex (35 days) and bisexually (28 days) Schistosoma mansoni-infected and Plasmodium berghei-immunized mice. Stereological analysis revealed that peak granuloma formation was already reached 8 days after injection in single-sex infected mice compared with 16 days in naive animals. No difference in granuloma formation between naive and P. berghei-immunized animals and between unisexually and bisexually S. mansoni-infected mice was observed. This suggests that the positive immunomodulatory effect on the granulomogenesis is worm specific and not likely to be due to arousal of the immune system by unrelated factors, nor is it influenced by the gender or degree of maturation of female worms. At all stages in time, the concanavalin A binding-fraction-induced granulomas reached only 65 to 70% of the volume of SEA-induced granulomas. Immunophenotyping of extracellular matrix proteins around deposited heads revealed that fibronectin was the dominant extracellular matrix protein and that also type I and IV collagen and laminin were deposited. Temporal analysis of the expression of the adhesion molecules ICAM-1, LFA-1, VLA-4, and VLA-6 was performed. Morphological evidence is presented for the role of adhesion molecules in the initiation and maintenance of hepatic granuloma formation. The chemokine monocyte chemoattractant protein-1 was expressed in the granuloma and in hepatic artery branches. From these data, it is concluded that adult S. mansoni worms positively modulate schistosomal hepatic granuloma formation in vivo. Adhesion molecules and chemokines play important roles in schistosomal granuloma formation.

Topics: Animals; Antigens, Helminth; Collagen; Concanavalin A; Cricetinae; Disorders of Sex Development; Egg Proteins; Fibronectins; Granuloma; Immunohistochemistry; Integrin alpha4beta1; Integrin alpha6beta1; Integrins; Intercellular Adhesion Molecule-1; Laminin; Liver Diseases; Lymphocyte Function-Associated Antigen-1; Male; Mice; Mice, Inbred Strains; Plasmodium berghei; Protozoan Proteins; Receptors, Lymphocyte Homing; Schistosomiasis mansoni; Sex Factors; Spleen

Schistosomiasis causes pathology in an estimated 200 million individuals. Clinical disease is caused by a complex immunopathologic response to the parasite ova, which are deposited in the host tissues. This immunopathologic response is caused by T lymphocytes which express the high-affinity IL-2 receptor (IL-2R). DAB389IL-2 is a diphtheria toxin-IL-2 fusion toxin protein which functionally inactivates or kills cells which bear the high-affinity IL-2R. DAB389IL-2 has been used in man to suppress IL-2R-dependent immune reactivity. Therefore, we reasoned that DAB389IL-2 might suppress immunopathology in schistosomiasis. In these studies we assessed the in vitro and in vivo effects of DAB389IL-2 on the development of immunopathology in murine schistosomiasis. DAB389IL-2 suppressed IL-2, lectin mitogen (Con A), and soluble Schistosoma mansoni egg antigen-induced lymphocyte proliferation and in vitro granuloma formation. In addition, DA-B389IL-2 suppressed in vitro IL-2R expression. DA-B389IL-2 also suppressed the development of granulomas and collagen deposition in vivo in the livers of infected animals. Therefore, DAB389IL-2 may have potential for the targeted reduction of immunopathology due to schistosomiasis in man.

Topics: Animals; Antigens, Helminth; Cell Division; Concanavalin A; Diphtheria Toxin; Epitopes; Granuloma; Immunosuppressive Agents; Immunotoxins; Interleukin-2; Liver Diseases, Parasitic; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Receptors, Interleukin-2; Recombinant Fusion Proteins; Schistosomiasis mansoni

Mice infected with Schistosoma mansoni mount focal granulomatous responses around each ovum that deposits in the liver and intestinal wall. The granulomas ultimately destroy the ova while absorbing the released toxic, injurious agents. The granulomas contain T cells and other cell types, all of which are under control. For example, T lymphocyte proliferation in situ within the granulomas is probably restrained by various regulatory mechanisms. Granuloma eosinophils make VIP, and granuloma T cells have VIP receptors. Yet, the function of VIP within the granulomatous response is unknown. We studied the effect of VIP on granuloma and splenic T cell proliferation in response to Con A or soluble egg antigens (SEA). [3H]Thymidine incorporation was used to assess the rate of proliferation. VIP decreased Con A- or SEA-induced, T lymphocyte proliferation. Suppression of proliferation was most evident for T cells stimulated submaximally with mitogen or antigen. Since T lymphocyte proliferation in response to antigen or mitogen requires soluble lymphokines, we investigated the capacity of VIP to alter the expression of several lymphokines as a possible mechanism for mediating suppression of T cell proliferation. VIP decreased IL-2 production, but did not effect IL-5 or IFN-gamma release. The effect of VIP on IL-2 production was dependent on the presence of a CD4+ T lymphocyte subset. VIP could no longer modulate lymphocyte proliferation if exogenous rIL-2 was added to the cultures. The addition of neutralizing anti-IL-2 mAb, but not anti-IL-4 mAb, substantially decreased granuloma lymphocyte proliferation in response to antigen or mitogen. This suggested that granuloma T cell proliferation required endogenously produced IL-2. These findings suggest that VIP may help modulate granuloma T cell proliferation through regulation of IL-2 production.

Topics: Animals; CD4-Positive T-Lymphocytes; Concanavalin A; Enterotoxins; Female; Granuloma; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred CBA; Recombinant Proteins; Schistosoma mansoni; Schistosomiasis mansoni; Spleen; T-Lymphocytes; Vasoactive Intestinal Peptide

In murine schistosomiasis, granulomas form around ova deposited in the liver and intestines of infected mice. The granulomas have eosinophils that produce vasoactive intestinal peptide (VIP) and T cells that display VIP receptors. IL-5 is a lymphokine important for the development and maturation of eosinophils. It seemed plausible that VIP, released from eosinophils, may interact with lymphocyte VIP receptors and modulate IL-5 production as part of a feedback regulatory circuit. Thus, we determined whether granuloma T cells make IL-5 and whether VIP modulates IL-5 production. Isolated granuloma cells enriched for T lymphocytes spontaneously released IL-5. Culture of these cells in the presence of VIP increased IL-5 secretion. Spleen cells were also studied. Spleen cells from infected mice did not spontaneously release IL-5 or express IL-5 mRNA and VIP did not stimulate these resting spleen cells to produce this IL. However, these cells did express IL-5 mRNA and secreted IL-5 in response to Con A or soluble egg Ag. VIP could not appreciably modulate IL-5 release when cells were cultured with VIP and the Ag or mitogen. Spleen cells washed free of Con A ceased IL-5 secretion within 24 h. These preactivated splenic T cells resumed vigorous IL-5 secretion in response to either Con A or VIP. Yet only Con A prominently induced IL-5 mRNA expression. VIP was an effective stimulus at concentrations equal to or above the kDa of the VIP receptor on both splenic and granuloma T cells (10(-8) M). It is concluded that, in murine schistosomiasis, VIP invokes IL-5 release from activated T cells that are not undergoing immediate TCR stimulation.

Topics: Animals; Antigens, Helminth; Concanavalin A; Eosinophils; Female; Gene Expression; Granuloma; Interleukin-5; Lymphocyte Activation; Mice; Mice, Inbred CBA; RNA, Messenger; Schistosomiasis mansoni; Spleen; T-Lymphocytes; Vasoactive Intestinal Peptide

T-lymphocyte-adherent mononuclear cell interaction was analyzed in the vigorous and immunomodulated liver granulomas of Schistosoma mansoni-infected mice. Collagenase-dispersed granulomas contained 15% lymphocytes, 30% macrophages, 50% eosinophilis, and some neutrophils. Dispersed granuloma cells stimulated with concanavalin A or soluble worm egg antigens (SEA) did not proliferate unless plate-adherent, esterase-positive mononuclear cells were removed before culture. To analyze the granuloma adherent cell-mediated suppression, vigorous granuloma cell cultures partially depleted of adherent mononuclear cells were supplemented with indomethacin, catalase, superoxide dismutase, levamisole, and anti-murine alpha/beta interferon antiserum. In concanavalin A and SEA-stimulated cultures, only the addition of indomethacin or anti-alpha/beta interferon antiserum alleviated the adherent cell-mediated suppression of vigorous granuloma lymphocyte response. In contrast, these agents only minimally alleviated the suppressed response of SEA-stimulated, immunomodulated granuloma lymphocytes. Moreover, coculture of equal numbers of vigorous and immunomodulated granuloma cells partially depleted of adherent suppressor cells abrogated the alleviated response of vigorous granuloma lymphocytes. These findings indicate that, within the schistosome egg-induced vigorous granulomas, the adherent mononuclear cells exert regulation over lymphocyte responsiveness by alpha/beta-interferon and an indomethacin-sensitive, probably prostaglandin-mediated pathway. Within the immunomodulated granulomas, the adherent suppressor cell-mediated regulation of lymphocyte proliferation appears to play a lesser role.

Topics: Animals; Antigens, Helminth; Concanavalin A; Granuloma; Immune Tolerance; Indomethacin; Interferon Type I; Leukocytes, Mononuclear; Liver Diseases; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred CBA; Schistosomiasis mansoni

Topics: Animals; Antibodies, Monoclonal; Cells, Cultured; Concanavalin A; Endothelium, Vascular; Granuloma; Immunohistochemistry; Inflammation; Lymphocytes; Lymphokines; Mice

Subcutaneous angioblastic lymphoid hyperplasia with eosinophilia (SALH) was reviewed with respect to eosinophil chemotaxis. Lymphoid cells separated from the granuloma spontaneously released at least two different eosinophil chemotactic factors (ECF): low-molecular-weight and high-molecular-weight ECF according to the profile on gel filtration (LMW-ECF, about 500; HMW-ECF, 45,000 to 70,000). The cells, however, failed to produce chemotactic activity for macrophages and neutrophils. By analysis with monoclonal antibodies against lymphocyte subpopulations, the granuloma T cells, probably OKT4-positive cells, were shown to be responsible for spontaneous production of these two ECF. Furthermore, the blood mononuclear leukocytes were separated from the patients with SALH. An ECF closely resembling HMW-ECF was also spontaneously produced by the blood OKT4-positive T lymphocytes, whereas no LMW-ECF was released. Mononuclear leukocytes from healthy donors, however, could produce an ECF resembling HMW-ECF and chemotactic activities for macrophages and neutrophils by stimulation with concanavalin A (Con A). Protein synthesis appeared to be essential for spontaneous ECF and for Con A-induced ECF production. These results suggest that the granuloma OKT4-positive T lymphocytes of the patients with SALH are in activated condition to release LMW- and HMW-ECF, whereas the blood OKT4-positive T lymphocytes are in activated condition to release only HMW-ECF. Such spontaneous and prolonged production of HMW-ECF by the cells can be one of the diagnostic means of SALH.

Topics: Angiolymphoid Hyperplasia with Eosinophilia; Antigens, Differentiation, T-Lymphocyte; Antigens, Surface; Chemotactic Factors; Chemotactic Factors, Eosinophil; Concanavalin A; Eosinophilia; Granuloma; Humans; Molecular Weight; Puromycin; T-Lymphocytes; Time Factors

Epithelioid cells from BCG-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas were examined for their ability to act as accessory cells for T-cell proliferation to mitogen (Con A) and antigen (PPD). The granuloma cells were separated on a FACS using monoclonal antibody specific to guinea pig macrophages. Epithelioid cells (which are Ia negative) were able to support proliferation to Con A but not to antigen. Cultures containing Ia positive granuloma macrophages from M. leprae sensitized animals did not show responsiveness to Con A or to PPD. Oil-induced peritoneal exudate macrophages from BCG or M. leprae immunized animals were able to act as accessory cells for both mitogen and antigen proliferation. The nonresponsiveness of cultures containing epithelioid cells stimulated with PPD or M. leprae granuloma macrophages stimulated with Con A was not due to suboptimal or supraoptimal accessory cell:lymphocyte ratios.

Topics: Animals; Antigen-Presenting Cells; Concanavalin A; Female; Granuloma; Guinea Pigs; In Vitro Techniques; Lymphocyte Activation; Macrophages; Male; Mycobacterium bovis; Mycobacterium leprae; Phagocytes; T-Lymphocytes; Tuberculin

Injections of appropriate numbers of irradiated tumor cells produced antibodies against tumor cell-surface antigen(s) in both syngeneic tumor models studied: the early transplant generations of the spontaneous L2 lymphoma in AKR/J mice and the chemically induced EL 4 lymphoma in C57BL/6J mice. No antibody was detected in normal or nonimmunized tumor-bearing mice. Tumor inhibitory or enhancing activity was not demonstrated by these antibodies. Immunoprophylaxis or cell-mediated immunity against the L2 lymphoma was not observed after injections of irradiated L2 cells and/or BCG into AKR mice. However, injections of irradiated EL 4 cells alone were effective in immunoprophylaxis against as many as 10(6) EL 4 cells and in immunotherapy against 10(2) EL 4 cells per mouse. The addition of BCG injections made immunotherapy with irradiated EL 4 cells effective against a load of 10(4) EL 4 cells/mouse, though BCG alone was not effective for immunoprophylaxis against EL 4 cells. Resistance to EL 4 could be transferred with viable syngeneic peritoneal or nucleated spleen cells. In both tumor models, an ongoing delayed hypersensitivity reaction to BCG alone apparently did not inhibit bystander tumor cells even when tumor cells were mixed before inoculation with viable BCG. In neither tumor model were concanavalin A-coated tumor cells more potent for immunoprophylaxis than were irradiated tumor cells alone.

Topics: Animals; Antibodies, Neoplasm; Antibody Formation; Antigens, Neoplasm; BCG Vaccine; Concanavalin A; Female; Granuloma; Immunity, Cellular; Immunization; Immunotherapy; Lymphocytes; Lymphoma; Mice; Mice, Inbred AKR; Mice, Inbred C57BL; Mycobacterium bovis; Neoplasms, Experimental