Eddie W. Lamon

Functional aspects of IgM and IgG Fc receptors on murine T lymphocytes.

All mammalian cells receive regulating signals via surface receptors of various kinds. These receptors specifically recognize molecules like hormones, transmittor substances, pharmacological reagents, mitogens and viruses. Lymphocytes have receptors for antigens (sig), activated complement components (C3receptors), various regulatory molecules released from cells activated by mitogens or viruses (e.g. interferon and TCGF), and finally receptors for the Fc part of immunoglobuiin molecules (FcR). FcR exist on a variety of different cell types where they may or may not be involved in the specific function of the cell. Among the most striking examples of FcR mediated cellular functions are Type I hypersensitivity mediated by mast cells via their FcR for IgF, transplacental transport of IgG, antibody-dependent cell mediated cytotoxicity (ADCC) by killer (K) cells, phagocytosis of antibody coated particles and modulation of immune responses (positive or negative) by immune complexes. The present review deals with studies on murine T lymphocytes bearing surface FcR. We will not limit the term receptor only to structures which mediate a functional change of the cells, but will rather use the term receptor

In vitro effects of retinoids on murine thymus-dependent and thymus-lndependent mitogenesis

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.

Potentiation of antibody responses by specific IgM: Specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.

Potentiation of IL-2-induced T-cell proliferation by retinoids☆

We evaluated the capacity of retinoids to potentiate proliferative responses of murine T-cells to recombinant human interleukin 2 (rIL-2). Concanavalin A (Con A) prestimulated spleen cells responded in a dose-dependent manner to added rIL-2. All-trans-retinoic acid (RA) at 10(-8) M potentiated the proliferative response by fivefold at saturating levels of IL-2. In similar experiments, two closely related retinamides, all-trans-(phenyl)retinamide (PR) and N-(4-hydroxyphenyl)retinamide (4-HPR), also potentiated murine splenocyte rIL-2 responses. Potentiation of IL-2-induced proliferation was dose-responsive to the concentration of added retinoid with peak potentiation occurring at 10(-10) - 10(-8) M in the presence of 10 U/ml rIL-2. Significant potentiation was observed at retinoid concentrations as low as 10(-14) M. Fluorescence flow cytometry of the responding cells revealed that among L3T4+, Lyt-2+ or total T-cells, at 72 h following Con A stimulation, essentially all of the cells expressed IL-2 receptors (IL-2R). This apparently represents near maximum IL-2R expression and treatment of the cells with retinoids did not increase IL-2R expression at that time point. The potentiation of IL-2 responses by retinoids was also observed with IL-2-dependent HT-2 cells, 98% of which were IL-2R positive. HT-2 proliferative responses to rIL-2 were potentiated as much as fourfold by 10(-10) M RA. HT-2 proliferative responses to rIL-2 were potentiated by all three retinoids dose dependently. Significant potentiation was observed with as little as 10(-14) M retinoid. Retinoids in the absence of IL-2 induced no proliferative responses. These data suggest that retinoids can augment the capacity of IL-2 to induce T-cell proliferation using Con A-activated murine splenic T-cell blasts and a long-term-cultured T-cell line.

In vitro effect of ethanol on cell-mediated cytotoxicity by murine spleen cells

The effects of ethanol on murine spleen cell-mediated lysis have been studied. Concentrations of 5.5-176 mM ethanol produced progressive inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC). Binding of spleen cells to antibody-sensitized target cells was not inhibited by comparable concentrations of ethanol. Kinetic analysis revealed decreased rates of lysis with increasing concentrations of ethanol. Changes of effector to target cell ratios revealed an inhibition of maximum lysis and decreased lytic efficiency in the presence of 88 mM ethanol. Preincubation experiments showed the inhibitory effect of ethanol to be reversible. Macrophage-depleted spleen cells appeared to be as susceptible to inhibition by ethanol as unfractionated spleen cells. Ethanol also inhibited natural killer and alloimmune cytotoxic T cell activity. The ADCC data were analysed by using a mathematical model which incorporates the kinetics of lysis, dose-response relationships, heterogeneity of the lytic effectors, reversibility of inhibition and ethanol loss during incubation. An inhibition constant (KI) of 373 mM-2 when two ethanol molecules interact with the site of inhibition was calculated. 50% inhibition of lysis is produced by 52 mM (0.24%) ethanol. The results are consistent with a model which assumes that lysis is due to a critical number of interactions which ultimately trigger the lytic event. Alcohol interferes with lysis by reacting with sites which are required for triggering the lytic event. Although the molecular details of the mechanism of inhibition are as yet undefined, we infer that ethanol inhibits ADCC at the programming for lysis or the lethal hit stages.

Effects of retinoids on human thymus-dependent and thymus-independent mitogenesis

The effects of all-trans-retinoic acid (RA), 13-cis-retinoic acid, and N-(4-hydroxyphenyl)retinamide on the mitogenic responses of various populations of human lymphocytes have been evaluated. Superoptimal concentrations of mitogens allowed the greatest RA-induced potentiation of lymphocyte proliferation. All three retinoids at concentrations as low as 5 x 10(-14)M significantly potentiated the proliferation of adenoidal and tonsillar lymphocytes stimulated by pokeweed mitogen (PWM). However, the responses of adenoidal and tonsillar lymphocytes to Staphylococcus aureus Cowan strain A were not potentiated by retinoids. Retinoids also caused significant potentiation of proliferation of PWM-stimulated peripheral blood lymphocytes (PBL). However, endpoint concentrations of retinoids required to significantly potentiate PBL proliferative responses to PWM were much higher than required for potentiation of adenoidal or tonsillar lymphocytes. PBL responses to concanavalin A (Con A) were significantly potentiated by retinoid concentrations as low as 10(-8) to 10(-10) M. Retinoid-potentiated responses were also observed wi Con A-stimulated thymocytes, but the endpoint concentrations required for significant potentiation were 10-fold higher than required to potentiate PBL responses to Con A. These data indicate that the sensitivity of lymphocytes to the retinoid-mediated potentiation of mitogenesis depends on the lymphoid compartment from which the cells are obtained. Tonsillar and adenoidal lymphocytes were the most responsive of the lymphocytes tested to the retinoid-induced potentiation of PWM responses. In addition, retinoids appear to selectively potentiate T cell-dependent proliferative activity.

Modulation of hapten-specific antibody responses with anticarrier antibody: I. Differential effects of IgM and IgG anticarrier on primary direct and indirect hapten-specific plaque-forming cells.

Summary Immunization of mice with TNP coupled to ORBC resulted in a TNP-specific antibody response. TNP-specific direct and indirect PFC in the spleens reached a maximum level 5 days after injection. Passively administered anticarrier antibodies injected with the hapten-carrier conjugates were found to modulate the primary PFC response to hapten. Three days after injection, both classes of carrier-specific antibody induced potentiation of hapten-specific direct and indirect PFC. On Day 5, however, IgM and IgG had opposite effects on the TNP-specific response. Carrier-specific IgM produced a three- to fourfold increase in both direct and indirect hapten-specific PFC over animals injected with the hapten-carrier conjugates alone. In contrast, carrier-specific IgG, at relatively high concentrations, suppressed the hapten-specific indirect PFC but had little effect on direct hapten-specific PFC.

Differential effects of retinoids on pokeweed mitogen induced B-cell proliferation vs immunoglobulin synthesis.

The effects of three retinoids, all-trans-retinoic acid (RA), 13-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) on mouse splenocyte responses to pokeweed mitogen (PWM) and Escherichia coli lipopolysaccharide (LPS) in vitro were evaluated. All three retinoids caused a dose dependent increase in the proliferative response to PWM. The retinoids hierarchy of efficacy based on potentiation of PWM-induced splenocyte proliferation was RA greater than cRA greater than 4-HPR. 13-cis-retinoic acid and 4-HPR also resulted in significant increases in Ig secretion in response to PWM. However, RA did not produce a significant increase in secretion compared with cells treated with PWM alone. The efficacy hierarchy of retinoids ability to potentiate Ig secretion was 4-HPR greater than cRA greater than RA. All three compounds did not affect Ig secretion from LPS-stimulated splenocytes and produced dose dependent decreases in proliferation. Both inhibition of LPS-induced proliferation and potentiation of PWM-induced proliferation were maximal when the retinoids were added during the first hour of culture. These results indicate that retinoids have a differential effect on Ig secretion and B-cell proliferation based on structural differences of the retinoids. Potentiation of proliferation and Ig secretion are both T-cell dependent and could be a result of increased lymphokine synthesis by the T-cells or increased responsiveness to the effects of the T-cell produced lymphokines.

Pharmacological and toxicological properties of arotinoids SMR-2 and SMR-6 in mice☆

Studies were conducted to define primary pharmacological and toxicological properties of two arotinoids, SMR-2 and SMR-6, in male B6D2F1 mice. Mice were gavaged daily for up to 22 days with retinoids in corn oil (0.1, 0.2, or 0.4 mg/kg day SMR-2 or SMR-6 or 2.5, 10, or 30 mg/kg all-trans-retinoic acid as a reference control). Toxicological and biochemical endpoints were assayed after 8, 15, and 22 days. At toxic doses, i.e., those inducing weight loss, morphological changes were observed in skin, lymph nodes, spleen, bone marrow, liver, thymus, forestomach, adrenal, bone, and testes. Biochemical alterations included elevated serum alkaline phosphatase, corticosterone, and interleukins-1, -2, and -3. Additional immune alterations included increased responsiveness of spleen cells to both thymus-dependent and thymus-independent mitogens and increases in the total number of B cells in the spleen. At doses not inducing weight loss, target organ effects included the appearance of plasma cells and infiltration of polymorphonuclear cells in lymph nodes; myeloid cell hypercellularity in bone marrow; hematopoiesis in spleen; subacute inflammation in forestomach; and periportal cytoplasmic vacuolization in liver. At the low doses, SMR-2 resulted in decreased responsiveness of spleen cells to mitogens and SMR-6 caused increased responsiveness. SMR-6 also increased interleukin-1 and-2 production at low doses. Biochemical effects included reduced activities of liver aryl hydrocarbon hydroxylase (AHH) and soluble brain protein kinase C. Overall, the results suggest that leukopoiesis and reduced liver AHH and reduced soluble protein kinase C activities are the primary and initial pharmacological and toxicological effects of retinoids.

Antibody-dependent cell-mediated cytotoxicity in the Moloney sarcoma virus system: No requirement for exogenous C5☆

Abstract It has been demonstrated that IgM antibody-dependent cell-mediated cytotoxicity (ADCC) does not require exogenous C5 in the Moloney sarcoma virus (M-MuSV) system by employing A J (C5 deficient) mice as a source of antibody, lymphocytes, and serum for support of cultures. The A J mice injected with the M-MuSV developed tumors within 5 days which were regressed by 16–20 days. The pooled antisera from these regressor animals were fractionated on Sephadex G-200 to obtain the 19 and 7 S fractions. The capability of the fractions to induce ADCC by spleen, lymph node, and thymus cells was tested against cells possessing the relevant virus-induced antigens and control cells lacking these antigens. The assays were carried out in medium supplemented with heat inactivated A J serum. The results indicate that C5-deficient A J mice are capable of regressing M-MuSV-induced tumors and, therefore, that exogenous C5 is not required for this in vivo response. These mice produce both IgM and IgG with very high titers in ADCC against the specific target cells. Lymphocytes from the spleen, lymph node, and thymus of A J mice are active effector cells against IgM-sensitized target cells with titers being generally higher with thymocytes. IgG-sensitized target cells were lysed only by splenocytes and lymph node cells. The involvement of membrane-bound complement components was not determined in this study. The assay was conducted in heat-inactivated serum in the absence of exogenous C5, and we conclude that IgG- and IgM-ADCC do not require the classical or alternative pathway of complement fixation.