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Calcium-independent phospholipase A2 (iPLA2) has been suggested to play an important role in the activation of caspase-1 induced by lipopolysaccharides (LPS). Here, we used pharmacological and genetic approaches to study the role of iPLA 2 in the activation of caspase-1. Bromoenol lactone (BEL), an inhibitor that was originally used to support a role for iPLA2 in the secretion of IL-1 beta, prevented caspase-1 activation induced by LPS and ATP as described, and also activation triggered by Salmonella infection and cytosolic flagellin, which rely on the Nlrc4 inflammasome. Analysis of BEL enantiomers showed that the S-BEL form was more effective than R-BEL in inhibiting the inflammasome, suggesting a role for iPLA2 . However, caspase-1 activation and IL-1 beta secretion and their inhibition by BEL were unimpaired in macrophages deficient in iPLA2 beta. BEL was originally identified as an inhibitor of serine proteases. Consistent with the latter, the serine proteases inhibitors TPCK, TLCK and AAF-cmk prevented the activation of the Nlrc4 and Nlrp3 inflammasomes while pan-cathepsin inhibitors were ineffective. These results indicate that iPLA2 beta is not critical for caspase-1 activation as currently proposed. Instead, the results suggest that serine protease(s) targeted by BEL may play a critical role in the activation of the inflammasome triggered by microbial stimuli.

We have previously reported that topical exposure of mice to oxazolone results in the appearance of regulatory mechanisms which markedly depress lymph node cell (LNC) proliferative responses to subsequent challenge with the same chemical. In the present study, we have sought to identify the cellular targets for such immunoregulation. Autoradiographic analyses revealed that although pre-exposure to oxazolone caused a substantial reduction of paracortical hyperplasia following challenge, the frequency of proliferating cells in lymphoid follicles was slightly increased. That B lymphocyte responses are unaffected by oxazolone-induced immunoregulation was confirmed by investigation of anti-hapten antibody formation by draining LNC. Challenge with oxazolone resulted in an accelerated antibody response in mice previously exposed to the same chemical. These data reveal that the active immunoregulation induced following sensitization with oxazolone is selective for T lymphocytes. Evidence is presented that CD4+ and CD8+ T lymphocytes possess equivalent sensitivity to these mechanisms.

2,4,6-Trinitrochlorobenzene (picryl chloride) and 2,4-dinitrochlorobenzene (DNCB) fail to cross-sensitize with respect to contact sensitivity in mice. Nevertheless, topical exposure of mice to DNCB and other skin-sensitizing dinitrobenzene derivatives was found to result in a significant impairment of draining lymph node cell proliferative responses induced following epicutaneous challenge with picryl chloride 5 days later. The inhibition of picryl chloride induced proliferation was associated with an impairment of contact sensitization to this chemical. The effect of DNCB on subsequent responses to picryl chloride was transient and no longer detectable 15 days following exposure. The inhibition of proliferation and contact sensitization caused by DNCB was largely restricted to picryl chloride. Thus, DNCB failed to influence the development of contact allergy to the unrelated chemical 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone) and exerted a far less pronounced effect on oxazolone-induced proliferative responses. These data, therefore, describe an antigen-restricted form of antigenic competition which is associated with a depression of the primary lymphocyte proliferative response.

The epidermis was stained with a panel of recently produced anti-guinea pig leucocyte antibodies. Guinea pig Langerhans cells were not detectable with antibodies directed against B lymphocytes (MSgp9), T lymphocytes (CT7 and MSgp7), T-helper/inducer (MSgp12) and putative T-suppressor/cytotoxic (CT6 and MSgp6) subsets. Langerhans cell expressed both major histocompatibility complex (MHC) class-I and II antigens and also an epitope (CT4) associated with lymphocyte migration, thus suggesting the migratory potential of this cell. Although the Langerhans cell did not express macrophage specific antigens, MSgp5, which detects lymphoid dendritic cells, was weakly expressed on the Langerhans cell. The Langerhans cell expressed a leucocyte-common antigen detected by H201. Double-labelling studies with anti-MHC class-II antibodies indicated that only 0.4 +/- 0.3% of the pan leucocyte-positive epidermal cells were Ia-negative, indicating that it is unlikely that a guinea pig analogue of the murine Thy-1 + dendritic epidermal cell (Thy-1 + dEC) exists.

A method for the quantitative measurement of macrophage aggregation by lymphokine preparations has been described previously. This involved the continuous measurement of the light absorbance of stirred suspensions of guinea pig peritoneal exudate cells (PEC). We now show that using a modified method, both aggregation of normal PEC, induced by preformed lymphokine, and direct aggregation of sensitized PEC with specific antigen can be measured. The method is suitable for the assay of large numbers of samples. Aggregation is shown to be immunologically specific for two antigens (bovine gamma-globulin and ovalbumin) and to be partially inhibited by sugars which inhibit migration inhibitory factor activity (rhamnose and fucose).

A comparison has been made of the 'flare-up' of 2,4-dinitrofluorobenzene (DNFB) contact reactions with that of 'flare-up' of potassium dichromate (K2Cr2O7) contact sensitivity reactions following the intravenous injection of dinitrobenzenesulphonic acid (DNBSO3) or K2Cr2O7. Differences in time course were noted and these were confirmed by increase in vascular permeability and histological appearance. Failure to influence the reactions with indomethacin would appear to exclude the participation of an Arthus-type phenomenon, despite evidence of a drop in the circulating antibody levels. Increased numbers of basophils were found particularly in the DNFB/DNBSO3 reaction during its resolution. These reactions could therefore be broadly included among the cutaneous basophil hypersensitivities.

The immunogenicities of two acrylate chemicals, trimethylolpropane triacrylate (TMPTA) and methyl acrylate (MeAc), and one related vinyl compound, 4-vinyl pyridine (4VP), were investigated by determining the in vivo induction of IgG antibodies in guinea pigs. The injection of the chemicals emulsified in Freund's complete adjuvant resulted in the induction of serum antibody responses against MeAc and 4VP but not TMPTA. However, antibody with anti-TMPTA activity was produced following immunization of guinea pigs with TMPTA conjugated to protein, which allowed comparisons to be made of the immunogenic structural features of the compounds.

Dendritic cells were enriched from guinea pig auricular lymph nodes containing Mycobacterium leprae induced granulomas by immunomagnetic depletion of other cells. These cells were strongly positive for major histocompatibility complex class II antigens and labelled with an antidendritic cell monoclonal antibody, but not with an antimacrophage antibody. Interdigitating dendritic cells were identified in the granulomatous lymph node by staining with the antidendritic cell antibody and by transmission electron microscopy. When cultured in vitro with purified T lymphocytes, these cells acted as accessory cells for both purified protein derivative and concanavalin A induced proliferation. Although previous studies have shown that macrophages from these lymph nodes do not act as accessory cells, the present results indicate that dendritic cells from M. leprae granuloma containing lymph nodes may act as antigen-presenting cells.

A mouse anti-guinea pig monoclonal antibody, designated MSgp 2, was derived by the fusion of NS-1 myeloma cells with mouse splenocytes hyperimmunized with guinea pig B cells. MSgp 2 reacts with an antigen present on the majority of lymphocytes. An unexpected finding was the expression of the MSgp 2 antigen on epidermal Langerhans cells, as defined by cell morphology, expression of MHC class II antigen and presence of Birbeck granules in positive cells. It is suggested that the tissue distribution of MSgp 2 antigen on lymphocytes of the lymph node could indicate a role for this determinant in cell migration.

Levamisole, an anti-helminthic drug used also as an immunostimulating agent, has been found to be a potent skin sensitizer in experimental animal models. Sensitization is associated with marked T-lymphocyte proliferation in the draining lymph node. It is suggested that immunostimulation through increase in the T-lymphocyte population could be a function of the strong sensitization induced by this compound.

A mouse anti-guinea pig monoclonal antibody, designated MSgp 1, was derived from a fusion between NS-1 myeloma cells and splenocytes hyperimmunised with guinea pig thymocytes. The MSgp 1 determinant is expressed by a subset of small thymocytes and lymph node T cells which participate in mixed leukocyte reactions. The determinant is modulated by antigen in vivo, and MSgp 1 antibody will prevent MHC class II driven proliferation in vitro. In addition, MSgp 1 reacts with a minor population of lymph node B cells, but not with a chronic B cell leukaemic cell line. Resident peritoneal macrophages express the MSgp 1 determinant, whereas chronic oil-induced peritoneal macrophages do not. The role of MSgp 1 defining guinea pig helper T cells is discussed by comparisons with other documented T helper cell reagents.

Heterologous antisera against guinea pig B lymphocytes have been prepared in rabbits. Sera, obtained 1 week after a third injection of cells from a guinea pig leukemic B cell line (L2C), were absorbed with guinea pig and sheep erythrocytes and guinea pig thymocytes. In the presence of complement, selected antisera killed both L2C cells (85%) and B cells from Hartley outbred guinea pigs (85%). Normal rabbit serum kills guinea pig thymocytes and this ability was enhanced in the sera obtained from rabbits immunized with L2C cells. After three absorptions with thymocytes the antisera retained their cytotoxicity for B cells but did not kill guinea pig thymocytes or peripheral T lymphocytes. The antigenic determinants on the L2C cells that simulate anti-B cell activity are unknown but surface membrane immunoglobulins were excluded. The development of methodology for obtaining a potent and specific anti-guinea pig B cell serum should significantly improve the usefulness of guinea pigs as animals for in vitro studies.

The ocular immediate hypersensitivity reaction in guinea pigs to topically applied normal rabbit serum can be evoked as long as 4 years after sensitization. The reaction was as severe and tended to persist for longer than that evoked 6 months after sensitization. Passive cutaneous anaphylaxis tests showed that very high titres of homocytotropic antibodies were present and that both IgE- and IgG1-like antibodies were involved. Sensitization with one set of injections instead of two was not consistently successful and the response on challenge was mild to moderate. Pretreatment of eyes with Isoptocarpine before antigenic challenge had no effect on the response. The addition of bacterial lipopolysaccharide to the first set of sensitizing injections produced hypersensitivity in animals which were otherwise refractory to sensitization.

Delayed type skin reactions in guinea pigs have been assessed by measuring three parameters: increase in skin thickness, diameter of erythema and intensity of erythema. Some groups of animals were immunized with different protein antigens in Freund's complete adjuvant with or without cyclophosphamide (CY) pretreatment; others received a single high dose of antigen intravenously at the time of immunization. The results emphasize the importance of measuring all three parameters for several days after skin testing. The intensity or erythema was found to be an especially useful parameter for assessing CY-induced modification of delayed hypersensitivity (DH) reactions. The increase in skin thickness, which can be measured objectively, was also valuable as both immediate and DH reactions are characterized by induration. The diameter of erythema could only be measured accurately for a short time after skin testing. Furthermore, the effects of intravenous antigen and CY pretreatment were not reflected by that parameter.

Intracheal intubation with the soluble metal salts potassium dichromate (K2Cr2O7) and nickel sulphate (NiSO4) causes a delay of up to 8 weeks in the development of delayed hypersensitivity to the specific agent. It is suggested that absorption of sensitizers by the respiratory route may, under certain circumstances, induce a state of specific immunological unresponsiveness, rather than necessarily lead to the development of a state of allergic sensitivity.

Guinea pigs immunized with DNP30-Ficoll produced IgM antibody only. No IgG1, IgG2, IgE antibodies or delayed hypersensitivity were detected in these animals. However, Arthus reactions, induced by the hapten coupled to a foreign carrier or the whole antigen, were found. The time course of the IgM response was limited and the response to reinjection of the antigen reduced. Cyclophosphamide (CY), given 3 days before primary immunization, prolonged the IgM response. Given on the day of immunization or 3 days after CY reduced this response. CY given on days +3 or +7 after primary immunization completely suppressed the response to antigen reinjected 42 days later. Arthus reactions were totally suppressed by CY given on the day of immunization, or 3 or 7 days later.

The expression of major histocompatibility (MHC) antigens on guinea pig T cells was used as a functional marker for lymphocyte activation. Antigen-stimulated lymphocytes were recovered from guinea pigs responding to the contact sensitizer DNFB, and isolated T cells were then phenotyped using a new antiguinea pig monoclonal antibody, MSgp7. The level of expression of MHC class II, as defined by the monclonal antibody, MSgp8, was increased on T cells recovered 4 days after sensitization, as compared with unsensitized controls. The value of this experiment was extended by measuring MHC class II expression on T cells stimulated in vitro by the mitogen concanavalin A, where a clear increase in MSgp8 binding was also observed. Confirmation of the specificity of MSgp8 for guinea pig MHC class II antigens was achieved by studying the inhibitory capacity of this antibody on an MHC class II restricted mixed leucocyte reaction. The combination of antibodies MSgp7 and MSgp8 with flow cytometry could be applied to other guinea pig experimental models to quantitate the expression of MHC class II antigens on T cells to determine their putative value in disease manifestation.