Marlene Aldo-Benson

The Association of Alcohol Consumption with Self-Reported Illness in University Students

Many reports over the years have indicated an association between alcohol consumption and infectious illness among chronic heavy drinkers; however, many patients in these studies have been chronically ill. Thus the question of whether alcohol can appreciably influence immunity in humans and affect the incidence of infectious diseases remains largely unanswered. For this study over 1,100 undergraduate students from a general education course at a large midwestern university were surveyed. Students were asked about their drinking habits and acute health problems. Analyses of their self-reports showed no increase in acute health problems or upper respiratory infections in students drinking between one and 21 drinks per week. However, students drinking 28 or more alcoholic drinks per week had significantly more health problems in the aggregate and those drinking more than 22 drinks per week had more upper respiratory infections than other students including nondrinkers. It was concluded that excessive alcohol intake increased the risk of respiratory infections and acute illnesses in these students, but more moderate alcohol consumption had little effect on the risk for these health problems.

SAA suppression of in vitro antibody response

In previous studies it has been shown that SAA isolated from serum of casein treated mice can suppress the in vitro antibody response of spleen cells to the T-dependent antigen sheep red blood cells (SRBC).’ This suppression of antibody response by SAA occurs in the early antigen recognition phase and not in the proliferative phase of the immune response? No effect upon the response of spleen cells to the T-independent antigen DNP-Ficoll was observed suggesting that SAA does not act directly on B-cells. Further studies were done to define the cellular mechanisms by which SAA exerts its effect on antibody response. These data suggest that SAA does not activate suppressor T-cells or cause induction of suppressor T-cells, but instead that SAA affects the interaction of macrophage and T-cells. In addition to these data from the murine system SAA has been shown to affect the response of human peripheral blood lymphocytes to SRBC.

Central suppression of monoclonal B cells: DNP-MGG suppresses proliferation and immunoglobulin synthesis in anti-DNP-secreting hybridoma and myeloma

Our laboratory has established that 2,4-dinitrophenyl-conjugated mouse IgG (DNP-MGG) can specifically suppress the anti-DNP secretion in hybridoma 35-12 and plasmacytoma MOPC-315 cells. To further study the mechanism of this suppression, the effect of DNP-MGG on anti-DNP synthesis and cell proliferation was investigated in these cell lines. Cultured tumor cells (1 X 10(6] were injected ip into syngeneic mice. These mice were then given either 1 mg MGG or 1 mg DNP-MGG. At different days after injection, tumor cells obtained from these mice were assayed for anti-DNP secretion, anti-DNP synthesis, cell proliferation, and tumor cell size. When the anti-DNP secretion was suppressed by DNP-MGG, the intracellular synthesis of anti-DNP, demonstrated by [3H]leucine incorporation into DNP-binding activity, was also suppressed. Simultaneous assays of [3H]thymidine incorporation demonstrated that proliferation was also suppressed. Tumor cells injected ip into mice normally become small nonsecreting cells and later return to preinjection size and secrete antibody. Those cells whose antibody synthesis and proliferation were suppressed by DNP-MGG remained smaller.

Tolerance and immunity in antigen-specific B-lymphocyte lines: Early receptor binding of either antigen or tolerogen initiates an immune response

Studies of the effect of tolerance-inducing compounds on B lymphocytes have been complicated by the fact that it is technically difficult to completely isolate the antigen-specific B cell from the effects of T cells or T-cell factors. We have used our cell lines of nonmalignant dinitrophenyl (DNP)-specific B lymphocytes derived from normal mice, which have no contaminating T cells, to study the effect of DNP-murine IgG2a (DNP-MGG), a tolerogen which is not normally immunogenic, on antigen-specific B lymphocytes. Preincubation with DNP-MGG for 48 hr, both in the presence and absence of T-cell factors from EL-4 supernatant prior to adding the antigen DNP-Ficoll, can induce tolerance in cell line B lymphocytes. The suppression is antigen-specific since preincubation with fluorescein-MGG or unconjugated MGG does not suppress the anti-DNP response. At least a 36-hr incubation is required for tolerance induction in B lymphocytes, but a 6-hr preincubation with DNP-MGG augments the immune response to DNP-Ficoll. Lymphocytes incubated for 6 or 24 hr with DNP-MGG prior to adding EL-4 supernatant and filler cells without DNP-Ficoll exhibited an immune response equal to that elicited by DNP-Ficoll and T-cell factors. A 6-hr pulse with a DNP-conjugated polymer of D-glutamic acid and D-lysine (DNP-dGL), a B-cell tolerogen which does not bind to Fc receptors, elicited the same immune response as seen with a 6-hr pulse of DNP-MGG but a 48-hr preincubation with DNP-dGL induced tolerance. Thus, it is likely that the initial binding of the tolerogen to the immunoglobulin receptor on the mature B cell elicits an activation signal similar to that seen with the antigen. The suppressive effect of the tolerogen itself appears to occur at a later stage of the process of the B-cell activation, proliferation, and differentiation.

Specific suppression of hybridoma immunoglobulin secretion by hapten-conjugated mouse IgG: A model of B-cell tolerance

In an effort to establish a model system for studying B-cell tolerance, the effects of hapten-conjugated isologous mouse IgG on the secretion of antibody by a mouse hybridoma cell line were studied. The hybridoma cell line 35-12 (HC) secretes IgM antibody to the hapten dinitrophenyl (DNP). After HC cells are injected intraperitoneally into BALB/c mice, the cells initially undergo a marked reduction in the proportion of PFC/10(6) followed by a return of PFC to pretransfer levels by 7-10 days. Hapten-conjugated mouse IgG (DNP-MGG), which induces tolerance to DNP in normal mouse B cells, also induces suppression of HC PFC when administered within the first 2 days after the transfer. Administration of tolerogen either 2 days before injection of HC or after the PFC response has returned to preinjection levels fails to give suppression. Suppression is dose dependent and hapten specific since immunogenic hapten-carrier conjugates (e.g., DNP-KLH, DNP-Ficoll) and fluorescein-MGG are not suppressive. T cells may not be required for suppression since hybridoma cells inoculated into nude mice are also suppressed by DNP-MGG. These results suggest that hybridoma cells undergo a change from nonsecreting to secreting cells during in vivo growth and that administration of tolerogen during the nonsecreting stage inhibits antibody secretion.

Membrane depolarization is induced in tolerant B lymphocytes by stimulation with antigen

Membrane depolarization is one of the earliest events in activation of cells by ligand receptor interaction. It is known that crosslinking of antigen-specific Ig receptors on B cells by antigen can induce membrane depolarization and subsequent Ia antigen expression on the cell surface. To determine whether a tolerance-inducing form of the antigen can also induce membrane depolarization after Ig receptor binding we used splenic B cells enriched for dinitrophenyl (DNP)-specific cells and determined relative membrane potential in these cells after binding of DNP-murine IgG2a (MGG) (tolerogen) or antigens (DNP-keyhole limpet hemocyanin (KLH) and DNP-Ficoll). Relative membrane potential was determined by loading the cells with the dye, 3.3-dipentyloxacarboxyanine (DiOC5(3)) after 2 hr incubation with ligand and determining relative fluorescence intensity on the fluorescence-activated cell sorter (FACS). Carriers alone did not depolarize these normal cell populations, but 100% of DNP-specific cells were depolarized by DNP-KLH and DNP-MGG while 85% were depolarized by DNP-Ficoll. To determine if tolerant B cells could be depolarized by antigen we induced tolerance in vitro or in vivo with DNP-MGG and measured the depolarization of DNP-specific B cells in response to antigens and tolerogen. DNP-specific B cells made tolerant by DNP-MGG underwent membrane depolarization when incubated with either DNP-KLH, DNP-MGG, or DNP-Ficoll but not with carriers alone. These data suggest that tolerogen induces membrane depolarization equally as well as antigen in normal cells. In addition, tolerant cells can be depolarized by Ig receptor crosslinking with either antigen or tolerogen. Thus, tolerance does not block the early membrane events induced by antigen in B cells.

Induction of suppressor T cells and tolerant B cells in vitro by DNP-IgG

Abstract Tolerance may be induced in vitro by preincubating cells for 6 hr with dinitrophenyl (DNP)-IgG prior to culturing with antigen, DNP-Ficoll, in Marbrook culture flasks. Studies were done to determine the mechanism of tolerance induction in this system. Isolated T cells and isolated B cells were preincubated either with or without DNP-IgG for 6 hr and placed in culture with antigen in various combinations. Tolerant T cells were able to suppress the anti-DNP response of normal B cells and normal spleen cells. Tolerant B cells and normal T cells also had a decrease in anti-DNP response. Tolerant T cells generated in vitro did not suppress the in vivo response of mice to DNP-Ficoll or DNP-KLH. These data suggest that in vitro tolerance induction by DNP-IgG is mediated by both suppressor T cells and tolerant B cells.

An in vitro model for clonal anergy in continuously growing antigen-specific B-cell lines☆

Two continuously growing nonmalignant B-cell lines specific for the hapten DNP have been used to study tolerance in developing B cells. These cell lines have previously been shown to consist of small cells without sIgM but with cytoplasmic mu chains, and mature sIgM- and sIgD-bearing cells. When the sIgM-negative cells are placed in culture, mature DNP-specific B cells begin to appear. The studies reported here have shown that when these cell lines were propagated in the presence of either 200 micrograms/ml or 1 mg/ml of the tolerogen DNP-MGG there was no inhibition of cell line growth as measured by thymidine incorporation, and no inhibition of receptor expression by maturing B cells. The cell line lymphocytes propagated in the presence of 200 micrograms/ml DNP-MGG for 7, 30, 45, or 60 days became tolerant and the tolerance persisted for at least 6 days after removal of DNP-MGG. However, tolerance was lost between 6 and 10 days after removal of DNP-MGG. Propagation of the cell lines for 30 days in either DNP-KLH or DNP-Ficoll produced the same results. Limiting dilution cultures of cell line lymphocytes made tolerant by growing them for 30 days in the presence of DNP-MGG demonstrated that there was a marked decrease in precursor frequency compared to controls. However, cell line lymphocytes made tolerant by a 48-hr incubation with DNP-MGG did not have a significant decrease in precursor frequency. These data suggest that tolerance induced by growing these cell lines in the presence of DNP-MGG is a valid in vitro model of tolerance in developing antigen-specific B cells. Tolerance induced in this model is consistent with the clonal anergy hypothesis, but requires the continued presence of DNP-MGG to maintain unresponsiveness. This suggests that clonal anergy can occur in B cells but may not be the sole mechanism of self tolerance for those antigens which are sequestered from the immune system.

Membrane dynamics of lymphocyte interaction with antigen and tolerogen.

The principle of hapten-specific carrier-dependent immunologic tolerance was used to study the in vivo and in vitro interaction of lymphocyte membrane receptors with antigen (DNP-KLH) and tolerogen (DNP-MGG). Direct fluorescent techniques were employed to illustrate the binding of tolerogeu and antigen to the same population of lymphoid cells and the subsequent in vivo and in vitro events related to capping and regeneration of membrane receptors.

Investigations of intrinsic abnormalities in DNA-specific B lymphocytes from autoimmune mice.

In murine models of systemic lupus erythematosus and in many humans with SLE, antibodies against native DNA (dsDNA) are a major contributor to the pathogenesis of the disease. Loss of self-tolerance to the DNA antigen may be associated with B-cell defects or regulatory cell dysfunction. We have developed B-cell lines with specificity for the antigen DNA, from both the autoimmune BWF1 mouse strain and from the non-autoimmune BALB/c strain, to use in the investigation of inherent B-cell defects in autoimmunity. Six BWF1 cell lines and five BALB/c cell lines which are free of Thy1.2+ cells and esterase positive cells, and have between 35 and 89% rosetting with dsDNA-SRBC targets, have been propagated in vitro for 24-36 months. The cells are non-malignant, growth-factor dependent and have no antigen or mitogen in the growth medium. Lyt-1 positive cells are found in the cell lines, but Lyt-1 negative cells are also present. They respond to the antigen DNA-HRBC when EL-4 supernatant is present in culture, and the peak of the plaque-forming cell (PFC) response is the same for both strains. When cells from both strains are cultured with varying amounts of T-cell factors, there is no difference in spontaneous antibody-forming cell (AFC) formation or in response to anti-mu stimulation between BWF1 and BALB/c strains. BALB/c spleen cells do not respond to DNA-HRBC in this culture system, but BWF1 spleen cells, as well as cell line cells from both strains, respond to this antigen. T cells from non-responding BALB/c spleen and responding BWF1 spleen are able to suppress the immune response to DNA-HRBC of cell line B cells from both strains. Propagating B-cell lines in the presence of DNA for 2 weeks stimulates BWF1 cell line cells, but suppresses the response of BALB/c cell lines to antigen.