Effects of suramin on the immune responses to sheep red blood cells in mice. II. In vitro studies

The effect of Suramin on the secondary in vitro response to sheep erythrocytes (SRBC) was studied. Spleen cells from mice which were treated with Suramin immediately prior to sensitization with SRBC failed to respond to an in vitro SRBC challenge. This Suramin-induced immunosuppression is not related to a defect in macrophage or B-cell function(s). Suramin does not interfere with the induction by SRBC of radioresistant and radiosensitive helper-T-cell subpopulations. Cell separation studies, using wheat germ agglutinin, showed radiosensitive helper-T-cell function in the nonagglutinated fraction while the radioresistant helper activities are carried out by the agglutinated subpopulation. Evidence is presented that Suramin administration results in a suppressive T-cell activity which can be demonstrated in the subpopulation agglutinated by wheat germ agglutinin. The role of such suppressive T cells in the inhibitory effect exerted by Suramin on the cell-mediated delayed-type hypersensitivity response to SRBC is discussed.     

The inducible costimulator plays the major costimulatory role in humoral immune responses in the absence of CD28

CD28 plays crucial costimulatory roles in T cell proliferation, cytokine production, and germinal center response. Mice that are deficient in the inducible costimulator (ICOS) also have defects in cytokine production and germinal center response. Because the full induction of ICOS in activated T cells depends on CD28 signal, the T cell costimulatory capacity of ICOS in the absence of CD28 has remained unclear. We have clarified this issue by comparing humoral immune responses in wild-type, CD28 knockout (CD28 KO), and CD28-ICOS double-knockout (DKO) mice. DKO mice had profound defects in Ab responses against environmental Ags, T-dependent protein Ags, and vesicular stomatitis virus that extended far beyond those observed in CD28 KO mice. However, DKO mice mounted normal Ab responses against a T-independent Ag, indicating that B cell function itself was normal. Restimulated CD4(+) DKO T cells that had been primed in vivo showed decreased proliferation and reduced IL-4 and IL-10 production compared with restimulated CD4(+) T cells from CD28 KO mice. Thus, in the absence of CD28, ICOS assumes the major T cell costimulatory role for humoral immune responses. Importantly, CD28-mediated ICOS up-regulation is not essential for ICOS function in vivo.     

Regulation of immune responses. I. Effects of cyclic AMP and cyclic GMP on immune induction.

Agents that raise intracellular cAMP levels (dibutyryl cyclic AMP, aminophylline, adenosine and butyric acid) increase the magnitude of an in vitro primary humoral immune response when added at 10^?3M during the first 12 hr of a 108 hr culture. Under the same conditions, cGMP has no direct effect but inhibits cAMP-mediated stimulation. DbcAMP (10^?3M or 10^?4M), present from 0 to 12 hr, also increases the number of cytotoxic lymphocytes in CBA/J (H-2^k) spleen cell cultures stimulated in a one-way mixed lymphocyte reaction with DBA/2J (H-2^d) spleen cells. The dbcAMP effect is antigen-dependent in both humoral and cell-mediated immunity and antigen-specific in the case of humoral responses.     

Spectrum of in vivo and in vitro cell-mediated immune responses in coccidioidomycosis.

The cell-mediated immune responses of 12 healthy, coccidioidin skin-test positive subjects (Group I) were compared with those of 15 healthy, coccidioidin skin-test positive persons who had primary asymptomatic coccidiodomycosis, (Group II), 12 patients with active, pulmonary coccidioidomycosis (Group III), four patients with disseminated disease (Group IV), and five patients who had been in clinical remission for 1 year or longer (Group V). Lymphocytes from healthy subjects in Groups I and II responded in vitro to Coccidioides immitis antigen by undergoing an increased DNA synthesis (lymphocyte transformation) and/or by producing macrophage migration inhibitory factor (MIF). In contrast, patients in Groups III and IV failed to respond to Coccidioides antigens in vivo (skin tests) or in vitro (lymphocyte transformation and production of MIF). The responses of subjects in Group V with inactive disease fell in between those of healthy donors in Groups I and II and patients in Groups III and IV. The cellular immune defect, in terms of antigen recognition, appeared to be specific for C. immitis in all but one patient.     

Cell-mediated immune responses in vitro. III. Elimination of specific cytotoxic lymphocyte responses by 3H-thymidine suicide.

The role of cellular proliferation in the development of cytotoxic lymphocyte (CL) responses in one-way mixed lymphocyte reactions was investigated by using tritiated thymidine of high specific activity to kill proliferating cells. To develop maximum CL responses, responding lymphoid cells must proliferate for approximately 72 hr; thereafter, precursors of CL appear to differentiate into active CL without further proliferation. Different alloantigen-sensitive precursor cell populations participate in the CL responses to each of two sets of stimulating alloantigens. When cells responding to one set of alloantigens were selectively destroyed after incorporating the hot thymidine, the surviving cells retained the capacity to develop a normal CL response to the second set of alloantigens.     

Hierarchical costimulator thresholds for distinct immune responses: application of a novel two-step Fc fusion protein transfer method

Activation of T cells is dependent upon coordinate engagement of Ag and costimulator receptors on their surfaces. In the case of the Ag receptors (TCRs), activation thresholds have been defined, with the number of TCRs that must be triggered to stimulate cytokine secretion by individual activated T cells differing for the various cytokines. In the present study, we have determined whether comparable activation thresholds exist for the costimulator receptors on T cells. To facilitate this type of quantitative costimulator analysis, we developed a novel two-step protein transfer approach that permits delivery of graded amounts of proteins to APC surfaces. By adding a human B7-1. Fcgamma1 (Fc domain of human IgG1) fusion protein to cells precoated with palmitated protein A, fine titration of the B7-1 extracellular domain was achieved. The B7-1. Fcgamma1 reincorporated into cell membranes by this method retained costimulator function, as measured by an in vitro proliferation assay. The degree of proliferation was dependent on the surface density of B7-1. Fcgamma1. Significantly, the threshold B7-1. Fcgamma1 density required for cytokine production differed between IFN-gamma and IL-2 and mirrored the hierarchy (IFN-gamma < IL-2) described previously for the TCR activation threshold. Hence, this study invokes a novel protein transfer strategy to establish that the levels of surface costimulator on APCs can dictate both the magnitude and the quality of evoked T cell responses. The notion of costimulator receptor activation thresholds emerges.     

Characterization of the effects of direct alkylators on in vitro immune responses

Although their mechanism of degradation may differ, both the SN1 alkylators, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-nitroso-N-methylurea (MNU), and the SN2 alkylators, dimethyl sulfate (DMS) and methyl methanesulfonate (MMS), spontaneously decompose under aqueous conditions to the methyldiazonium ion or a direct methylating intermediate, respectively. Thus, these agents serve as useful probes to investigate the immunosuppressive potential of the putative primary reactive intermediate of dimethylnitrosamine (DMN) metabolism, the methyldiazonium ion. The effects of these direct alkylating agents on the in vitro immune response were characterized. Direct addition of both the SN1 and SN2 alkylators to naive B6C3F1 murine splenocytes produced a dose-dependent suppression of the in vitro antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocytes (sRBC), T-independent antigen, dinitrophenyl (DNP)-Ficoll, and the polyclonal activator, lipopolysaccharide (LPS). The T-dependent and T-independent responses proved to be more sensitive than the polyclonal response to the effects of these compounds, except for MNNG in which all 3 antibody responses were equally affected. The suppression of the AFC response for all antigens was unaffected by the addition of 2-ME, and was observed at concentrations below those affecting viability, although at the highest concentrations an effect on viability was often observed. The addition of MNNG to the T-dependent AFC response at any time within the first 96 h produced a marked suppression, while the addition of DMS to cultures was only effective in suppressing the AFC response if added within the first 24 h. MNNG and DMS suppressed the proliferative responses to both B-cell (LPS) and T-cell (Concanavalin A; Con A) mitogens, as well as in the mixed lymphocyte response (MLR). In addition, a positive correlation between immunosuppression and DNA damage, as measured by single-strand breaks, was observed. Although these compounds produced suppression of in vitro immune responses, their profile of activity on immunocompetence and DNA damage was different from that associated with DMN and thus, the direct alkylators may not prove to be useful models to elucidate the mechanism of the DMN-induced immunosuppression.     

Different individual immune responses elicited by in vitro immunization

We have previously demonstrated that the addition of muramyl dipeptide (MDP), interleukin-2 (IL-2) and IL-4 effectively raises antibody production from L-Leucyl-L-leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBLs) against specific soluble antigen when immunized in vitro. However, PBLs from individual donors were separate optimal conditions regarding concentrations for IL-2 and IL-4, which in turn required us to optimize each individual PBLs to effectively produce antigen specific human antibody by in vitro immunization. These individual differences in the requirement for IL-2 and IL-4 reflects the differences in individual immune responses against a specific soluble antigen, which can be elicited by in vitro immunization. In the present study, we investigated these individual differences in the requirement for IL-2 and IL-4 to induce antibody productionin vitro in the PBLs of 12 volunteers (9 healthy donors and 3 allergenic patients). IL-2 requirements for antibody production varied dependent upon each donor, while higher amounts of IL-4 inhibited IgM and IgG production in all of the healthy donors. However, some of the characteristic features for PBLs donated from allergenic included lowered IgM production compared to PBLs derived from healthy donors, and very high IgE production in the absence of cytokines and allergen. These results demonstrate that the sensitivity of PBLs against antigen sensitization differs between healthy donors and atopic patients, which suggests that the frequency of antigen sensitization might be reflected in differing activation states and/or differing subpopulations of lymphocytes in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro anamnestic immune responses and modulating factors

Summary The term anamnestic refers to the specific and enhanced immune responses of antigen-immunized (primed) lymphoid memory cells to secondary challenge with a foreign substance (antigen). These responses include the accelerated and quantitatively greater syntheses of antibody and other macromolecules than upon primary challenge of such cells. Rabbits were primarily immunized with keyhole limpet hemocyanin (KLH). Six days later their memory lymph node cells (LNC) were removed, and upon culture with KLH, responded with the synthesis of antibody, immunoglobulin (Ig), protein, DNA and RNA, as well as with active transport of dibutryl cyclic AMP (DbcAMP). Purified thymus-derived (T) LNC were prepared on anti-rabbit Ig affinity columns. Bursal-equivalent (B) cells were prepared by binding to a complex of sheep erythrocytes (SRBC)-antibody to SRBC-complement and centrifugation of these complexes on suitable gradients. When these T and B KLH-primed LNC were mixed and challenged with KLH the aforementioned macromolecular syntheses and active transport occurred. Indeed, by a variety of criteria, the reconstituted anamnestic immune responses were indistinguishable from these responses of unfractionated LNC. Antigenic stimulation of KLH-primed T cells induced the synthesis of proteins and DNA, but not antibody, but antigenic challenge of KLH-primed B cells did not evoke these syntheses. However, added KLH induced a mixture of T and B antigen-primed LNC to synthesize more protein, Ig, DNA than either population alone and more antibody than T cells per se; B cells required help for all of these responses. The thymus (T) cell-dependent phase of in vitro anamnestic antibody response lasted the first 24–36 hr.The antibody response was regulated by antigen-concentration. One g KLH evoked maximal antibody synthesis, 10 and 100 g KLH much less. Challenge of the separated T and B cell populations with different KLH concentrations, followed by recombination and eventual assay of antibody synthesis revealed different optima. The optimal concentration for T cell help was 0.01–0.1 g KLH; higher amounts induced much less antibody production. The optimum for B cells was 1–10 g KLH; 100 g inhibited antibody formation.The antibody response to KLH and human serum albumin (HSA) was regulated nonspecifically utilizing LNC from rabbits immunized simultaneously with these two antigens. Thus stimulation of LNC from these rabbits with either antigen induced the synthesis of antibodies to both antigens. HSA and KLH did not cross-react either serologically or cellularly. Cross-stimulation of antibody synthesis also was observed when rabbit LNC were primed with KLH and Mb. However, in this instance, cross-reaction between KLH and sperm-whale myoglobulin (Mb) was observed at the cellular, presumably the T cell, level, although not at the antibody (B cell) level. The antibody response could also be modulated by exogenous cholera enterotoxin (CT), dibutyryl cyclic AMP (DbcAMP) and prostaglandins of the E series. The addition of each substance together with 1–100 g KLH to KLH-primed LNC enhanced the antibody response many-fold. CT-induced non-immunized LNC to produce soluble factor(s) (SF) which, when added to KLH-primed LNC together with KLH, enhanced antibody synthesis significantly. The addition of Indomethacin, an inhibitor of PGE synthesis to KLH-immunized cells together with KLH inhibited antibody production, suggesting that PGE was involved in this response. Evidence was adduced that neither cyclic AMP nor PGE was required for the antibody response: Ca²⁺ was not required for induction of this response by KLH, but only its regulation by cAMP.Moreover, when KLH-primed LNC were fractionated on Nylon columns, the effluent cells were induced by KLH to synthesize antibody, but this synthesis was not enhanced by added DbcAMP or PGE; presumably, regulatory cells were removed on the column. Added KLH induced PGE synthsis in these cultures; this synthesis required macrophages. In all of the LNC cultures — including cultures from rabbits immunized with KLH, HSA, and MB months or a year earlier — much antibody synthesis occurred even when antigen was not added to the cultures. This spontaneous antibody was anamnestic, thymus (T cell)-dependent and involved the interaction of residual immunogen on dendritic cells with T and B memory cells. This spontaneous antibody response provides a model for the study of the factors involved in the longterm maintenance of humoral immunity.Mb was employed as a source of more refined antigenic determinants. Rabbits were immunized with Mb in complete Freunds adjuvant. The addition of small synthetic peptides corresponding to the five antigenic sites of Mb to the Mb-primed LNC induced the synthesis of antibody, Ig, protein, DNA, RNA, and macrophage migration inhibitory factor (MIF). The N terminal 1–6 peptide, which is not antigenic, i.e. does not combine with antibody to Mb, also induced all of these syntheses, except MIF. These peptide-induced responses appeared to be thymus-dependent.Abbreviations AP alum-precipitated - AFab goat IgG antibody to rabbit Fab - ATG goat IgG antibody to rabbit thymocytes - BGG bovine gamma globulin - Bsa bovine serum albumin - BAC bromo acetyl cellulose - B bursalequivalent lymphocytes - CT cholera enterotoxin - CRL complement receptor lymphocytes - DFA complete Freund's adjuvant-, - cAMP adenosine 3:5-cyclic monophosphate - cGMP guanosine 3:5-cyclic monophosphate - DbcAMP N⁶,O²-dibutryl cyclic AMP - EAC sheep erythrocytes sensitized with antibody and complement - FITC fluorescein isothiocyanate - HSA human serum albumin - KLH keyhole limpet hemocyanin - LNC lymph node cells - MEM minimum essectial Eagle's medium - medium; MIF m crophage migration inhibitory factor - Mb sperm-whale myoglobin - PHA phytohemagglutinin - PGE prostaglandins of the E series - PGF prostaglandins of the F series - PGSI inhibitors of prostaglandin systhesis - Slg surface immunoglobulin - T thymus-derived lymphocytes     

Human immune responses to hapten-conjugated cells. II. The roles of autologous and allogeneic histocompatibility determinants in proliferative responses in vitro.

Proliferative responses of human lymphocytes primed in vitro to autologous TNP-cells were found to be associated with autologous D-region determinants irrespective of HLA-B locus antigens. Family studies of secondary TNP-conjugate proliferative responses demonstrated a gene dosage effect in this phenomenon. Moreover, co-culture with allogeneic cells did not affect the net TNP-conjugate proliferative responses of primed responder cells, suggesting that HLA-D region preference was due to a requirement for representation of TNP-molecules in association or combination with autologous MHC structures. Alloantigens were found to influence the sensitization of lymphocytes to autologous hapten-conjugated cells. Co-culture of allogeneic and TNP-modified autologous stimulator cells in primary cultures enhanced the secondary TNP proliferative response. Sensitization of human lymphocytes to allogeneic cells alone did not prime responses to autologous modified cells. However, priming lymphocytes to modified autologous cells potentiated responses to allogeneic cells. The data suggest a complex relationship between responses to alloantigens and modified autologous cells.     

Cell-mediated immune responses in vitro. II. Simultaneous generation of cytotoxic lymphocyte responses to two sets of alloantigens of limited cross-reactivity.

The conditions for generation of simultaneous and independent cytotoxic lymphocyte (CL) responses to each of two sets of alloantigens of limited cross-reactivity by mouse spleen cells in vitro have been investigated. Responder spleen cells were incubated with mitomycin C-treated C57BL/6 (H-2b) or DBA/2 (H-2d) stimulator spleen cells and day 5 CL responses were assayed with 51Cr-labeled EL-4 leukemia (H-2b) and P815 mastocytoma (H-2d) as target cells. Spleen cells from mice of the various H-2 haplotypes tested differed greatly in their ability to develop specific CL responses against alloantigens on the stimulator spleen cells and in the degree of cross-reactive cytotoxic activity against target cells bearing alloantigens not present on the stimulator spleen cells. In contrast to the other strains examined, DBA/1 (H-2q) spleen cells developed specific CL responses to either H-2b or H-2d alloantigens without exhibiting significant cross-reactive activity on the inappropriate target cell. The CL responses to H-2b and H-2d alloantigens by DBA/1 spleen cells were comparable in magnitude and had similar stimulator cell-dose requirements. Further, DBA/1 spleen cells developed CL responses of normal magnitude simultaneously against both target cells when incubated with both mitomycin C-treated C57BL/6 and DBA/2 stimulator cells.     

Effects of marrow donor and recipient age on immune responses

This report describes treatments to restore diminished splenic immune responses of old mice. Lethal irradiation, followed by young bone marrow and infant thymus transplants, restored the T cell mitogen response and the antibody-forming cell response against sheep red blood cells in the old mice. Although old bone marrow cells restore these immune responses in young recipients, as well as do young bone marrow cells, old bone marrow in old recipients did not improve their levels of response. Longevities of old recipients with rejuvenated responses were not increased, and aging of tail tendon collagen was not affected. The effect of lethal irradiation before the marrow transplant was shown to be minimal, by the use of unirradiated old W-anemic recipients. Parabiosing young mice with old partners caused impairment of these two immune responses in the young partners without enhancing them in the old partners. The old partners did not have increased longevities. To explain these results, we suggest the following hypothesis: old bone marrow contains precursors that produce suppressive factors or cells when in an old environment but not when in a young environment. However, these factors, if allowed to develop in an old environment, can function in a young parabiosed partner.     

Influences of roxithromycin on cell-mediated immune responses.

We investigated the effects of roxithromycin (RXM), a synthesized macrolide antibiotic on murine cellular immune responses by examining the in vitro proliferative response of lymphocytes, interleukin 1 (IL-1) production and interleukin 2 (IL-2) production. RXM was orally administered to BALB/c mice at a dose of 5 mg/kg once a day for 42 days. Spontaneous blastic activity of lymphocytes prepared from mice administered with RXM for 7 days was higher than those from control mice. The activity peaked at the 14th day, and then decreased gradually to control levels by the 42nd day. Time kinetics of lymphocyte blastogenesis to concanavalin A showed a pattern similar to that observed in spontaneous blastic activity. Oral administration of RXM also influenced cytokine production; short-term (for 14 days) administration of RXM enhanced both IL-1 and IL-2 production but long-term (for 42 days) administration inhibited them.     

Effects of two bioflavonoids on certain cellular immune reactions in vitro

Oxidative and autoaggressive immune processes are supposed to be involved in the pathogenesis of certain chronic liver diseases. In this study the effects of two naturally occurring antioxidant flavonoids, (+)cyanidanol-3 and silymarin, were determined on T and active T cell percentages, antigen-dependent (ADCC), lectin-dependent (LDCC) and natural (NK) cell mediated cytotoxicity and on lectin induced blast transformation of lymphocytes from healthy subjects and patients with chronic alcoholic liver disease in vitro. We observed no effects on T and active T cell percentages and on ADCC activity. Both drugs decreased LDCC activities of patients and lectin-induced lymphoblast transformation of controls and patients, (+)cyanidanol slightly and silymarin significantly decreased NK activities of controls and patients. These suppressive effects could partly be explained by the free radical scavenger and lipoxigenase inhibitor activity of the drugs and support the promising role of bioflavonoids in the treatment of chronic liver diseases.     

Effects of two lipid lowering therapies on immune responses in hyperlipidemic subjects

Aims

To compare the effects of two of the most effective lipid-lowering therapies with similar LDL-cholesterol reduction capacity on the innate and adaptive immune responses through the evaluation of autoantibodies anti-oxidized LDL (anti-oxLDL Abs) and electronegative LDL [LDL(−)] levels.

Main methods

We performed a prospective, randomized, open label study, with parallel arms and blinded endpoints. One hundred and twelve subjects completed the study protocol and received rosuvastatin 40 mg or ezetimibe/simvastatin 10/40 mg for 12 weeks. Lipids, apolipoproteins, LDL(−), and anti-oxLDL Abs (IgG) were assayed at baseline and end of study.

Key findings

Main clinical and laboratory characteristics were comparable at baseline. Lipid modifications were similar in both treatment arms, however, a significant raise in anti-oxLDL Abs levels was observed in subjects treated with rosuvastatin (p = 0.026 vs. baseline), but not in those receiving simvastatin/ezetimibe. (p = 0.233 vs. baseline), thus suggesting modulation of adaptive immunity by a potent statin. Titers of LDL(−) were not modified by the treatments.

Significance

Considering atherosclerosis as an immune disease, this study adds new information, showing that under similar LDL-cholesterol reduction, the choice of lipid-lowering therapy can differently modulate adaptive immune responses.