Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae in mice.

Traditionally, adjuvants have been administered with antigens to enhance immunity. We studied the effect of several adjuvants such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS), homopolymers of polyinosinic-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U), lithium chloride (LiCl), saponin Quil A and calcium phosphate gel (CaHPO(4)) on the immune response of mice to formalin-inactivated Mycoplasma agalactiae. The specific antibody or cytokine producing splenocytes were detected by ELISAspot and immunocytochemistry, respectively. Depending on the adjuvant given, the number of M. agalactiae-specific antibody producing cells was increased 2.5-6-fold. IgG was the major class of M. agalactiae-specific antibodies followed by IgM, IgA and IgE. Among IgG isotypes, FCA, FIA, Quil A and CaHPO(4) induced an IgG1 response with substantial increase of the IgG2a, IgG2b and IgG3 isotypes while poly I:C shifted the response toward an IgG2a/IgG3 production. Finally, poly A:U induced an IgG2b response while LPS and LiCl augmented the IgG3/IgG1/IgG2a secretion. FCA augmented IL-4, IL-5 and IL-10 production suggesting a strong Th2 response, while IFN-gamma and IL-12 remained low; poly I:C enhanced IFN-gamma, IL-12 and TNF-alpha eliciting a Th1 response; poly A:U resulted in a IL-10, IL-5, IL-6 and IL-12 secretion; and LPS enhanced the IL-10, IL-6 and TNF-alpha production. Our data show that adjuvants augment M. agalactiae-specific antibody production and lead to B cell isotype-switching via the appropriate cytokine milieu. Certain adjuvants, such as poly I:C, therefore, appear as promising immune enhancers for vaccination against M. agalactiae infections.

The anti-inflammatory effect of opioids.

The anti-inflammatory activity of two novel opioids PM and PO as well as of pethidine was studied. The mouse paw edema, induced by various phlogistic agents, was significantly inhibited after the administration of opioids, fact that was independent of their antioxidant properties. The anti-inflammatory action of the above opioids was not reversed by naloxone. These results suggest that a variety of complex regulatory activities may be performed by opioid agonists via naloxone-sensitive or naloxone insensitive receptors on inflammatory cells, directly or indirectly by the inhibition of cytokines and mediators involved in inflammation.

Modulation of alpha 1-acid glycoprotein (AGP) gene induction following honey bee venom administration to adjuvant arthritic (AA) rats; possible role of AGP on AA development.

Honey bee venom (HBV) administration to adjuvant arthritic (AA) rats resulted in a significant suppression of arthritis and in suppression of the hepatic acute phase alpha 1-acid glycoprotein (AGP) gene induction at the early stages of disease development. AGP administration in AA rats resulted in acceleration of arthritis development and in increase of severity and duration of the disease. IL-1, IL-6, tumour necrosis factor (TNF) and glucocorticoids alone are not responsible for the HBV-mediated AGP gene down-regulation. These results indicate that AGP gene expression in AA and HBV-treated AA rats involves the interaction of several factors, and that AGP plays a role for AA development in rats.

Suppression of adjuvant-induced disease (AID) by a novel analgesic-opioid agonist which also possesses antioxidant activity (neuroimmunomodulation).

A centrally acting analgesic-opioid agonist, PM, suppresses the primary inflammation and the secondary lesions of adjuvant-induced disease in rats. This compound also possesses antioxidant properties and inhibits the carrageenin-induced edema in mice. PM, therefore, is interfering to immune functions probably by binding to opioid receptors on lymphocytes and to inflammatory cells by its antioxidant activity.

Restoration of impaired drug metabolism of rats with adjuvant induced disease by two steroids with different influence on drug biotransformation.

Adjuvant Induced Disease (AID) produced by intradermal administration of Freund's Adjuvant to Fisher female rats, caused inflammation and severe impairment of the drug metabolic activity of the liver. Treatment with pregnenolone-16 alpha-carbonitrile (1), or triamcinolone (2) caused a mild and a great reduction of the produced arthritis, respectively, while these steroids completely restored the established drug metabolic impairment of the AID rats. It is concluded that: (i) There is a cross-linkage between arthritis and liver function, the effect of the former to the latter is greater than vice versa. (ii) The action of 1 on the impaired hepatic drug metabolic activity is direct, while that of 2 is indirect. (iii) The effect of the two steroids on AID and liver drug metabolism is not mediated via a protective action on lipid peroxidation.

Interleukins counteract opioid agonists immunosuppression.

A centrally acting novel analgesic, PM, added to murine lymphocyte cultures abrogated the mitogenic response to Con A, as well as, interleukin 2 production, in a dose-dependent manner. Simultaneous presence of interleukin 1 (IL-1) and interleukin 2 (IL-2) into the cultures counteracted, in a dose-dependent manner, as naloxone does, the immunosuppressive action of PM as well as of Pethidine, a known opioid agonist. These results show that IL-1 and IL-2 together exhibit classical agonist-antagonist opioid receptor interactions and support the hypothesis that these lymphokines might play a role as endogenous opioid receptor antagonists against endogenous opioid agonists.

Regulation of isotype immunoglobulin and interleukin production by adjuvants in vitro.

An in vitro system has been developed in which antibodies to fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) or dextran sulfate (DXS), are produced in the presence or absence of different adjuvants. The antibody response of in vitro cultures was measured by assaying the total Ig-secreting cells and FITC-specific plaque-forming cells (PFC). The presence of low levels of antigen and various cytokines were necessary for the production of isotypes other than IgM. Our results indicate that the regulation of isotype switching in vitro is dependent upon the non-specific stimuli-adjuvants, which probably activate different types of cells for cytokine production. The pre-activation of spleen cells, in our system, by antigen, seems to play a decisive role in the subclass of IgG produced. The adjuvant may still be able to influence the precommitted cell isotype to switch to another subclass but only in a "down-stream" direction i.e., IgG3----IgG1----IgG2b----IgG2a.

Regulation of isotype immunoglobulin production by adjuvants in vivo.

Mice were immunized against fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) or dextran sulphate (DXS) in the absence or presence of different adjuvants. The immune response was assayed as the total Ig-secreting cells and FITC-specific plaque-forming cells (PFC) found in various lymphoid organs. The adjuvants influenced the isotype of antibodies produced to the same antigenic determinant. The PFC of different IgG subclasses were favoured by different adjuvants. The IgG3 isotype was produced mainly after immunization with either antigen and lipopolysaccharide (LPS) or Li salt as adjuvant; IgG1 was produced with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), alum, poly I:C, Quil A, Be salt, and poly A:U. Some of the above adjuvants (Be salt and poly A:U) favoured the production of IgG2b, and others (CFA, alum, Quil A, and poly I:C) favoured the IgG2a isotype besides the main isotype. Attempts were made to correlate the activation by the various adjuvants of certain TH subtypes with the isotypes produced.

Immunosuppression by a novel analgesic-opioid agonist.

The effect of a new, centrally acting analgesic, 2-n-pentyloxy-2-phenyl-4- methyl-morpholine (PM) on the humoral antibody isotype responses, mitogenic responses, and interleukin production and assay was studied. Treatment with this opioid agonist exerted a suppressive effect on the antibody responses to TD [sheep red blood cells (SRBC), fluoresceinated human gamma globulin (HGG-FITC)] and TI [fluoresceinated dextran (DEX-FITC), lipopolysaccharide (LPS)]antigens in mice. The suppression was found to be dose- and time-dependent for all antigens tested, suggesting that PM affected both T and B cells. PM impaired lymphocyte functions, as the in vitro T and B mitogen reactions were inhibited in a dose- and time-dependent manner in mice and rats. PM, even at the highest concentration used, could not completely inhibit the production of interleukin 1 (IL-1)-like activity, but it caused complete inhibition, in a dose-dependent manner, of the production of IL-2-like activity. In addition, PM inhibited the assays of both IL-1 and IL-2. Naloxone counteracted all immunosuppressive effects of PM in vivo and in vitro. From this it was concluded that PM operates on the immune system directly, via opioid receptor mechanisms. Our data suggest that immunosuppression by PM, an opioid agonist, may be exerted by an inhibition of interleukin action on lymphocytes, and they confirm the important role of opiate receptors in lymphocyte function.

Interleukins production and responsiveness in Fisher rats with adjuvant-induced disease: role of suppressor cells.

Splenic macrophages from AID Fisher rats produce normal levels of IL-1, in contrast to IL-2 which is significantly lower than normal. A state of hyporesponsiveness of spleen cells to Con-A and PHA occurs with similar kinetics to and is correlated with the development of an articular arthritis; IL-1 addition to AID spleen cell cultures does restore to normal their deficient response, in contrast to addition of IL-2 or rIL-2. The spleen cells from normal or AID rats are equally sensitive to the inhibitory effects of suppressor glass adherent cells found in AID spleens on lymphocyte proliferation. Our results indicate that these suppressor cells interfere with IL-1 production or IL-1 binding to T-cells and that this interference is not due to passive absorption of interleukins by these cells.

Effect of magnesium deficiency on interleukin production by Fisher rats: effect of interleukins on reduced in vitro lymphocyte responses to concanavalin A and lipopolysaccharide.

The ability of rats fed a magnesium-deficient diet to produce interleukins (ILs) and the effect of ILs on in vitro lymphocyte mitogenesis have been studied in rats. Lack of magnesium resulted in a lower number of plastic-adherent spleen cells and in a reduction of IL-1 production. IL-2 production was not significantly affected, indicating differential sensitivity of T cells to magnesium deficiency. The diminished mitogenic response of splenocytes to concanavalin A (Con-A) was restored by the addition of IL-1 supernatant, while the addition of IL-2 supernatant and recombinant IL-2 resulted in significantly greater enhancement of proliferation in response to Con-A, compared with that of control spleen cells. The fact that recombinant IL-2 restored the T-cell responses to Con-A indicates that the active factor in the IL-2 supernatant is IL-2.

Bee venom, adjuvant induced disease and interleukin production.

Interleukin production and the in vitro mitogenic responses from honey bee venom treated normal rat splenocytes were reduced considerably compared to controls. Addition of interleukin-1 (IL-1) or interleukin-2 (IL-2) supernatants to these cultures in vitro resulted in an increase of their responses to normal levels. These results suggest that in vivo honey bee venom treatment affects the production of IL-1 by macrophages directly. Honey bee venom treatment affects adjuvant induced disease development by inhibiting certain macrophage functions and thus indirectly inhibiting the activation of T and B cells, and possibly the activation of an endogenous virus which might be involved in adjuvant induced disease induction.

Effect of age on the induction of in vitro drug metabolism by pregnenolone-16 alpha-carbonitrile (PCN); effect of age and PCN on immune responses.

The drug metabolizing capacity and the immune responses of normal and PCN treated young, adult and old rats were studied. In normal young and old rats the drug metabolism in general and the immune responses were reduced in comparison to adult animals. PCN treatment caused significant increase of drug metabolism in all age groups of animals due to induction of the microsomal enzymes of the liver. However, there were certain drug dependant variations in the different age groups of rats. PCN given in vivo affected differently the responses of T and B-cells in the young, adult and old animals. PCN is a non-hormonal microsomal enzyme inducer and the question a-rises as to whether there is any connection between this process and the immune system.

Restoration of immunosuppression in lung cancer by normal sera.

The ConA and PWM responses of blood lymphocytes from lung cancer patients (LC) was found to be diminished in comparison to normal. LC sera inhibited the blastogenic response of normal human and rat lymphocytes, suggesting that there is a common inhibitory mechanism. Addition of normal serum to lung cancer blood lymphocytes allows a restoration of the diminished proliferative responses to normal or near normal levels, except to high ConA concentrations. Possible explanations for this finding are discussed.

Adjuvants influence the immunoglobin subclass distribution of immune responses in vivo.

Mice were immunized against fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) in the absence or presence of different adjuvants. The immune response was assayed every other day with regard to both total Ig-secreting cells and FITC-specific plaque-forming cells (PFC). The adjuvants influenced the type of immune response induced to the same antigenic determinant. Thus, addition of Freund's complete (FCA) or incomplete (FIA) adjuvant preferentially led to the secretion of IgG1 PFC of an average high affinity. Most newly appearing IgG-secreting cells were also detected as FITC-specific PFC. The use of lipopolysaccharide (LPS) as an adjuvant resulted in the induction of both IgM and IgG, particularly of the IgG3 and IgG2b subclasses. However, these antibodies had relatively low affinity, and a large number of total IgG-secreting cells induced by LPS had no detectable FITC specificity. The FCA/FIA- and LPS-induced responses to FITC-HGG were additive when injected together, indicating that they act on distinct subpopulations of B lymphoid cells. The adjuvant response to LPS, but not the response to FCA/FIA, was totally absent in mice of the C3H/Hej strain, which are non-responders to the polyclonal activating properties of LPS. Finally, the response induced by FCA or FIA was T-cell-dependent and the LPS response T-cell-independent as assayed in nude mice.

Effect of diet on adjuvant-induced disease and mitogenic responses of Fisher rats.

Fisher rats from a inbred colony, when fed on a salt-free high-protein diet, developed only a mild arthritis after adjuvant injection. Their spleen cells failed to respond in vitro to concanavalin A (a T-cell mitogen), although they possessed a B-cell function of plaque formation to sheep red blood cells. When a full salt supplement was included in the diet, or magnesium or copper or zinc was included in the drinking water, adjuvant-induced arthritis was severe and the response to the T-cell mitogen was restored. The above results suggest that these trace elements may stabilize or activate certain cell populations needed for some immune responses in rats.

Diminished lymphocyte responses to phytohemagglutinin in lung cancer.

The PHA response of blood lymphocytes from lung cancer patients was found to be diminished in comparison to normal. Sera from these patients inhibited the blastogenic response of blood lymphocytes from normal subjects. Normal sera could restore to various levels the diminished PHA response of lymphocytes from lung cancer patients. The results suggest that the immunosuppression seen in lung cancer may be mediated by a factor (s) in the serum which might bound reversibly to a certain subpopulation of T-cells and permanently to another and or some other inhibitory mechanism does exist.