Mechanism of Enhanced Humoral Response in Rodents by a Synthetic Polymer

Abstract

We previously reported that administration of a low molecular weight (MW=800) synthetic polymer, NED 137, significantly increases humoral and cellular immune responses in the rat. The effect of NED 137 on the murine humoral response to T-dependent (TD) and T-independent (TI) antigens was studied in C57 BL/6, CBA/J and Balb/c mice. The TD antigens (SRBC, DNP-DA with adjuvant) or TI antigen (DNP-Ficoll) were administered simultaneously with NED 137. The polymer significantly increased the direct PFC response to all antigens tested in normal mice. However, it could not restore the PFC response to SRBC in athymic (nu/nu) mice. The effect of NED 137 on accessory cells was studied by the assessment of the in vitro response to SRBC in normal and macrophage-depleted rat spleen cultures. The polymer stimulated both, the primary and secondary IgM response and its immunopotentiating activity was the greatest in macrophage-depleted spleen cell preparations. The lack of effect of NED 137 in systems devoid of functional T cells, dependency on and specificity for a sensitizing antigen and its ability to stimulate a secondary response suggest that this polymer does not act as a “conventional” B-cell polyclonal activator.     

Mechanisms of Altered in Vitro Antibody Response in the Rat by a Small Synthetic Polymer

NED 137, a low molecular weight synthetic polymer, enhances the rat humoral immune response to cellular antigens in vivo. The effect of NED 137 on the in vitro antibody response to sheep erythrocytes was studied in normal and macrophage-depleted rat spleen cultures. The polymer stimulated the IgM antibody response of normal splenocytes, and its immunopotentiating activity was increased in macrophage-depleted spleen cell preparations. NED 137 enhanced both the primary and the secondary plaque-forming cell (PFC) response of rat splenocytes in a dose- and time-dependent manner. This stimulatory effect of the polymer was dependent on the presence of antigen and demonstrated antigen specificity. The PFC and mitogenic responses of both normal and macrophage-depleted rat splenocytes were dramatically inhibited in cultures prepared from animals pretreated with NED 137 before culture initiation. The antigen dependency, specificity and potentiation of a secondary response suggest that NED 137 acts through the triggering of the lymphoid system but not as a 'conventional' B-cell polyclonal activator.     

Selective immunosuppression resulting from exposure to the carcinogenic congener of benzopyrene in B6C3F1 mice.

B6C3F1 mice were exposed to two congeners of benzopyrene, either the carcinogen benzo(a)pyrene (B(a)P) or the non-carcinogen benzo(e)pyrene (B(e)P. Exposure of mice to B(a)P resulted in a reduced number of IgM and IgG antibody plaque forming cells (PFC) to the T-dependent (TD) antigen SRBC and IgM PFC's to the T-independent (TI) antigen LPS. The IgM response to hapten conjugated TI antigens was examined using TNP-LPS for reactivity of less mature B cells (B1) and TNP-Ficoll for more mature B cells (B2). Exposure to B(a)P severely depressed the TNP-Ficoll PFC response by up to 77% without altering the TNP-LPS response. These data indicated that exposure to B(a)P alters differentiation and antibody production in mature B cells to both TD and B2 TI antigens. No change in PFC was observed following exposure to B(e)P. Mishell-Dutton co-cultures confirmed that B cells were affected and that T helper cells or suppressor Mphi were not involved. Parameters of cell-mediated immunocompetence including delayed cutaneous hypersensitivity to KLH, allograft or tumour cell rejection and susceptibility to Listeria monocytogens were unaltered in B(a)P treated mice.     

The allogeneic effect in (Balb/c X AKR)F1 mice

Graft-versus-host reaction induced in 6-weeks-old (Balb/c X AKR)F1 hybrid mice by the injection of parental (Balb/c) spleen cells caused the allogeneic effect, i.e. the stimulation of host IgM and IgG antibody forming cells (PFC) to SRBC and LPS. Parental lymphocytes in vivo allosensitized to AKR antigens were used to find out if the alloreactive T lymphocytes mediate the allogeneic effect. It was found that these cells influenced differently the humoral response to the antigens. The stimulation of the response to LPS was not changed in comparison with that produced by normal parental cells. On the 5th day of immunization with SRBC the stimulation of IgM PFC was stronger than IgG PFC. On the 10th day, the stimulation of IgM and IgG PFC was decreased. Moreover, it was observed that when parental splenocytes normal or allosensitized were incubated and centrifuged on allogeneic fibroblast monolayer the ability of nonadherent cells to mediate allogeneic effect was decreased or completely disappeared.     

Impaired antibody response against T-dependent antigens in rhino mice.

The antibody response in rhino mice, which carry a mutant gene hrrh, to thymus-dependent (TD) or thymus-independent (TI) antigens was compared with that of phenotypically normal littermates. The magnitude of antibody response to TD antigens in rhino mice decreased as they grew up, whereas the antibody response to TI antigens in rhino mice was indistinguishable from that of littermates. A transfer of thymus cells from littermates to rhino mice resulted in the partial restoration of the responsiveness to TD antigens. The experiments employing adoptive transfer of spleen cells from rhino mice to the irradiated normal mice suggested that the hyporesponsiveness of TD antigens of adult rhino mice was mainly due to the defect in the T helper cell activities rather than either the increase of the suppressor cells or defects in other cell types.     

Properties of antisera against lymphocytes of nude mice.

This paper reports results of a study on the activity of rabbit antisera against nu/nu Balb/c lymphocytes in vivo and in vitro. It was found that ALS against nu/nu lymph node cells suppressed the alloantigen reaction and the sRFC or PFC formation for T-dependent (SRBC) and T-independent (LPS) antigens. ALS against nu/nu spleen cells affected only the sRFC and PFC for T-independent antigen. The former serum exhibited a high cytotoxicity for the suspensions enriched or depleted in B cells while the latter one was more cytotoxic for the suspension enriched in B cells. It indicates that ALS anti nu/nu spleen cells is specific for B lymphocytes and ALS anti nu/nu lymph node cells is directed not only to B cells but also to a subpopulation of T lymphocytes. It suggests the existence of a subpopulation of T lymphocytes in nu/nu lymph node cells.     

Change in the Humoral Response of Athymic Nude Mice with Ageing

The evolution of the serum Ig levels of Balb/c-nu/nu mice was investigated between 1 month and 12 months of age. An increase as a function of age was observed in all classes and subclasses, which was, expressed in percentage of a nu/+ serum, from 130% to 230% for IgM, from 3% to 24% for IgG1, from 12% to 164% for IgG2a, from 28% to 62% for IgG2b, and from 10% to 50% for IgA. This increase correlates with an increase of plasma cells of each class in the bone marrow, whereas the number of plasma cells in the spleen, the lymph node, and the intestinal mucosa did not change markedly with age. The humoral response after an injection of heterologous erythrocytes was compared in young and aged nu/nu mice; aged mice had a higher haemagglutination titre mainly due to direct (IgM) antibodies. The response of the spleen, as judged by plaque-forming cells (PFC), was similar in young and aged mice, but the bone marrow response, not detectable in young mice, was about as high in aged nude mice as in nu/+ mice. Although the content of Thy 1 cells in the spleen and lymph nodes was markedly higher in aged than in young nude mice, no T-cell function could be detected at any age, either in the response to phytohaemagglutinin or concanavalin A or in a graft-versus-host assay. Increase in the Ig production with age is interpreted as the result of progressive priming and hyperimmunization by environmental antigens, leading to a T-independent immune response (even against antigens considered to be T-dependent) predominantly located in the bone marrow.     

Cellular aspects of non-specific stimulation of antibody production by capsular polysaccharide of Klebsiella pneumoniae

Comparative studies were made of the increase in the numbers of plaque-forming cells (PFC), rosette-forming cells (RFC) and haemolytic foci for erythrocyte antigens in the spleens of mice given a non-specific stimulus (the capsular polysaccharide of Klebsiella pneumoniae (CPS-K)) and an antigenic stimulus (sheep red blood cells (SRBC)). The number of direct PFC for SRBC was increased by injection of CPS-K to as high a level as that obtained by injection of SRBC. In contrast, by injection of CPS-K the numbers of indirect PFC, RFC (probably the antibody-forming cell precursors) and haemolytic foci were not increased significantly, whereas all of them were increased markedly by injection of SRBC. The maximal number of PFC in mice injected with CPS-K was approximated to the number of background RFC of the same mice. Injection of CPS-K generated 25–130 times more direct PFC for each of three kinds of erythrocyte antigens, SRBC, rabbit red blood cells and chick red blood cells, than background PFC, whereas the total number of spleen cells was not increased significantly or increased very slightly. Repeated injections of CPS-K were not significantly more effective for increase in the number of direct PFC than a single injection of CPS-K. Injection of CPS-K could generate many direct PFC in mice which had been thymectomized, irradiated and reconstituted with foetal liver cells. In mice injected with CPS-K, increase in (or maintenance of) the numbers of direct PFC and RFC were inhibited by injection of a mitogen inhibitor, vinblastine sulphate, but their sensitivities to the drug were less than those found in mice immunized with SRBC. It has been concluded from these results that in mice injected with CPS-K a large number of antibody-forming cell precursors are differentiated to direct PFC through one division or a few divisions of the individual cells, and that the inability of CPS-K to induce sufficient cell divisions of the individual precursor cells is the cause of the lack of increase in the number of indirect PFC and in immunological memory for secondary PFC responses in mice injected with CPS-K.     

Dissociative effects of malarial infection on humoral and cell-mediated immunity in mice.

The effect of malarial infection on immune responses was studied in mice. When sheep red blood cells (SRBC) were injected 2 days before or at the same time as infection with Plasmodium berghei, there was a marked increase in the number of splenic plaque forming cells (PFC) induced by SRBC as compared with uninfected controls. When SRBC were injected 2 days or more after the infection, however, the PFC response was significantly reduced. On the other hand, cell-mediated immunity, as exemplified by delayed-type hypersensitivity (DTH) to a number of antigens, was suppressed whether the infection was introduced before or after antigen stimulation. A similar effect could be produced by injecting the host with the supernatant obtained following incubation in vitro of peripheral blood from heavily infected mice. When this supernatant was injected i.v. into normal mice at the same time as SRBC priming, it enhanced the humoral response to SRBC, but suppressed the DTH to SRBC. The coincident induction of this inverse relationship between humoral and cell-mediated immunities was clearly borne out by a dose response study using different dilutions of supernatant. The active component appeared to be of large molecular weight (greater than 150,000), thermostable and not present in the serum of infected mice.     

Differential effects of nordihydroguaiaretic acid (NDGA) on B-cell subsets: reversal of NDGA-induced antibody suppression by cyclic GMP is subset specific

Nordihydroguaiaretic acid (4,4'-[2,3-dimethyl tetramethylene]-dipyrpcatechol) (NDGA), a reportedly specific lipoxygenase inhibitor, suppressed the in vitro murine plaque-forming cell (PFC) response to a thymus-dependent (TD) antigen, and the two subclasses of thymus-independent (TI) antigens, TI1 and TI2, at a final concentration of 33 microM. Suppression kinetics were inconsistent with those observed in previous experiments for other lipoxygenase inhibitors, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), in that BHA and BHT exert suppression early in the 5-day PFC culture system, whereas NDGA suppressed through the 96th h of the 120 h culture period. The sulfhydryl-protective agent 2-mercaptoethanol (2ME) protected both the TD and TI responses. Dibutyryl cyclic GMP (dbcGMP) failed to restore NDGA-suppressed TD and TI1 PFC responses but restored the TI2 response when added at 0-, 24-, 48-, 72-, and 96-h at concentrations of 1-2 mM. NDGA inhibited lipopolysaccharide- (LPS-) induced increases in murine splenocyte cyclic GMP (cGMP) levels, and dbcGMP failed to accelerate the onset of the TI2 PFC response appreciably. The results of these and other laboratory studies indicated that NDGA may not be a specific inhibitor of lipoxygenase. Furthermore, the B-cell subset responding to TI2 antigens may be separated from the TD- and TI1-responding subsets because of the ability of dbcGMP to restore NDGA-suppressed TI2 responses but not the TD or TI1 response. The results suggest a fundamental difference in the biochemical pathways of B-cell subsets, and that cGMP metabolism in some cells may be linked to specific protein synthesis.     

Immunomodulation by Trichinella spiralis: primary versus secondary response to phosphorylcholine-containing antigens

The present work was designed to determine in detail the capacity of the nematode Trichinella to modulate the plaque-forming cell (PFC) response of BCF1 mice to the parasite's own antigens. To this end, we studied the PFC responses shown by infected and non-infected BCF1 mice using as the target antigen phosphorylcholine, an epitope which is found in the parasite. From the results presented here, the following conclusions can be drawn: i) Trichinella spiralis is capable of modulating the immunoresponse to thymus-dependent (TD), but not to thymus-independent (TI), parasite antigens; ii) Trichinella spiralis suppresses the PFC response to the parasite-derived TD antigen FCp1 (a particulate antigen containing PC) during the muscle stage of its life cycle, but does not affect the responses to other parasite-derived PC-bearing antigens; this seems to indicate that the suppressive activity exerted by Trichinella is highly specific; iii) anti-PC PFC production in the secondary response was also suppressed by the parasite. Finally, the inability of the FCp1 antigen to induce detectable anti-PC PFC, other than IgM, is discussed.     

Regulation of immune response to SRBC: suppressor cell activity induced by soluble fraction of antigen.

Water soluble fraction (SF) of SRBC was obtained by hypotonic lysis and ultracentrifugation. SF was found to induce very weak SRBC-specific antibody response in mice. Pretreatment with SF accelerated direct PFC response to SRBC and accelerated and suppressed indirect PFC response. Spleen cells from mice treated with SF exhibited enhanced response to SRBC after transfer in irradiated recipients. The transfer of spleen cells from mice treated with SF to normal non-irradiated mice markedly suppressed the recipients' PFC responses to SRBC. The observed suppressive effect is interpreted as a consequence of suppressor cell activity.     

The potentiality of antibody-producing cells. I Bispecific cell occurrence in double stimulated cultures of syngeneic or allogeneic spleen cells of the mouse.

Cultures of spleen cells from Swiss, C57Bl or DBA/2 mice stimulated with 2,4,6-trinitrophenyl (TNP) conjugated sheep erythrocytes (SRBC) were used for studying the in vitro responses to TNP and native SRBC antigens and the frequency of occurrence of cells responding to both antigens. The response was revealed by plating the cultured cells with both native SRBC and TNP-conjugated pigeon erythrocytes (TNP--PRBC). Specific responses were obtained in all the cultures. Bispecific haemolytic plaque-forming cells (PFC) were detected in almost all cultures of individual Swiss mice cells with a frequency of 2-5--14 PFC/10(6) cells recovered. In DBA/2 cell cultures bispecific PFC were found in half the cultures (2-5--8-3/10(6) cells) and in C57Bl cell cultures in 30 per cent of the cultures (7--21/10(6) cells). When cells from individual Swiss mice immunized in vivo with TNP--SRBC were as an allogeneic culture from the 2nd day after immunization in the presence of TNP--SRBC, the frequency of bispecific PFC increased from 8 to 30/10(6) cells. Mixed allogeneic cultures of normal C57Bl and DBA/2 cells yielded high specific responses with regular occurrence of bispecific PFC only when the numbers of cells cultured together was small. However, when allogeneic cells were mixed 24 hours after starting the cultures, all responses were stimulated and bispecific PFC were found in considerable numbers (4--33/10(6) cells). Cross-reactivity between TNP--PRBC and native SRBC antigens was studied by culturing cells with each of the antigens and plating the cells with both, or by immunizing in vivo with SRBC or PRBC and culturing the cells with both antigens from the 2nd to the 5th day after immunization with both antigens. In no instance did bispecific PFC exceed background levels (0-1--0-6/10(6) cells) in these control experiments.     

Unresponsiveness following immunization with the T-cell-independent antigen dextran B512. Can it be abrogated?

The bacterial carbohydrate dextran B512 is a thymus-independent (TI) antigen and a poor immunogen. Humoral responses consist primarily of IgM and no memory response is observed; rather, secondary responses to native dextran are similar to or suppressed compared with primary responses. However, immune responses to dextran can be enhanced. In this study we have used a protein–dextran conjugate that elicits a thymus-dependent (TD) immune response against dextran. Furthermore, we used the potent adjuvant cholera toxin (CT) for the dextran immunizations. This enables us to re-evaluate the phenomenon of poor secondary response to dextran and whether it can be abrogated. We show that native dextran-primed mice were not able to mount IgG anti-dextran antibody responses after repeated immunizations with the TD, protein–dextran conjugate. This was also apparent in the spleen, where almost no dextran-specific germinal centres were detected. However, the anti-protein antibody response was normal in these mice, demonstrating that it is only the anti-dextran-responding cells that are affected. The effect of CT adjuvant on these events was also evaluated. CT enhanced the humoral IgM anti-dextran responses as well as the splenic responses to dextran. But, the isotype profile was not altered, still no IgG was produced. In contrast, mice primed with the TD conjugate and repeatedly re-immunized with native, TI, dextran generated IgG anti-dextran responses. Our results indicate that it is probable that the lack of proper costimulation in the initiation of the response to dextran causes the suppressed secondary dextran responses. Furthermore, these results suggest that TI and TD forms of dextran activate the same type of B cells, since TI dextran-priming abrogated TD dextran IgG responses. The importance of the priming event for the induction of a classical memory response to carbohydrate antigens and the implications for vaccination strategies, are discussed.     

Effect of 1-thiocarbamoyl-2-imidazolidinone on the generation of plaque forming cell responses

Although 1-thiocarbamoyl-2-imidazolidinone (TCI) is a highly potent modulator of cellular immunity, its effects on humoral immunity have not been investigated. Given orally to mice prior to immunization with sheep red blood cells (SRBC), low TCI doses (10(-14) to 10(-10) g/kg) suppressed primary plaque forming cell (PFC) responses of spleen cells by 50-75%. TCI effects in vivo were dependent on drug dose, antigen dose and time of drug administration relative to immunization. The kinetics of this response were not appreciably altered by TCI. Higher TCI doses, immunization with higher levels of SRBC than required to produce a maximal response or administration of TCI later than 48 hours after immunization resulted in drug effects ranging from slight suppression to mild enhancement of the primary PFC response. TCI given in vivo enhanced primary PFC responses to the T-independent antigen DNP-Ficoll by greater than 700%+. TCI given before a primary immunization suppressed a secondary PFC response to SRBC elicited 28 days later. However, when TCI was given 24 h prior to a secondary immunization, doses greater than 10(-5) g/kg were necessary to suppress the PFC response. The effect of TCI on in vitro immunized spleen cell cultures was similar to that found for in vivo immunized mice. TCI at concentrations up to 10(-1) g/l did not cause a loss of lymphocyte viability or inhibit plaque production by antibody producing cells. Effects of TCI on PFC responses in vitro were reversible if cells briefly exposed to an optimal concentration of drug were washed extensively prior to immunization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Mechlorethamine on SRBC-Induced Secondary Antibody Response in Mice

Abstract

The effect of low-dose mechlorethamine (5μg/kg) on secondary humoral response to sheep red blood cells (SRBC), depending on time of exposure to the drug in relation to priming and challenge was studied in Balb/c mice. It was found that mechlorethamine in a dose of 5 μg/kg stimulated primary humoral response to SRBC resulting in the increased number of the plaque forming cells (PFC) and hemagglutinin titre (19S + 7S). However, this effect waned 10 days after immunization. On the other hand, the same mechlorethamine dose potentiated secondary humoral response to SRBC and increased the number of PFC and anti-SRBC hemagglutinin titres (notably 7S), which was due to the challenging antigenic stimulus. In each immunization, mechlorethamine administration prolonged the potentiating effect of the drug on anti-SRBC hemagglutinin titre. When mechlorethamine was administered to the mice only after priming, the number of PFC increased, but anti-SRBC hemagglutinin titre (7S) remained unchanged. This was likely due to the fact that mechlorethamine administered after priming increases the number of long-lived lymphocytes B, which in turn affect secondary humoral response.     

Distinction between suppressors of the delayed-type hypersensitivity and the humoral response to sheep erythrocytes.

Suppressors for both delayed-type hypersensitivity (DTH) and the humoral immune response could be simultaneously induced in the spleens of mice by immunization with a high dose of SRBC. Normal recipient mice of the spleen cells from donors immunized 5 days previously elicited depressed DTH or humoral response when immunized with SRBC. The suppressive activity was found to reside in T not B enriched fraction. Four hundred rad irradiation of the primed spleen cells resulted in complete loss of DTH suppressor activity, but only in some reduction of the suppressor activity for the humoral response. In contrast, hydrocortisone treatment of the donor mice caused no loss of DTH suppressor activity while approximately half of the suppressive activity for anti-SRBC PFC response was lost. Adult thymectomy prevented completely the induction of the DTH suppressor in contrast to little loss of the suppressor activity for the humoral response. DTH suppression was antigen-specific for the induction, but nonspecific for the expression. However the suppression of the humoral response was antigen-specific not only for the induction but also for the expression. In addition, DTH suppressor was capable of suppressing both the induction and expression of DTH while the humoral response was suppressed only in the induction stage by the suppressor.     

The immune response in NZB mice of different ages to thymus-dependent and thymus-independent phosphorylcholine antigens.

This study was designed to examine aberrations of immune responses in autoimmune NZB strain mice during ageing, at the level of individual B cell clones. The response to phosphorylcholine (PC) was chosen because murine responses to PC are restricted to a few B cell clones and can be characterized with idiotypic markers. Responses to both thymus-dependent (TD) and thymus-independently (TI) PC-containing antigens were measured in mice ranging from 1 to 62 weeks of age. We found that: (1) TD responses to phosphorylcholine keyhole limpet haemocyanin (PC-KLH) decreased markedly (about 17-fold) between 28 and 62 weeks of age. TI responses to the R36a strain pneumococcus decreased only slightly during the same period. (2) The PFC responses to both antigens became markedly prolonged in mice older than 24 weeks. (3) The NZB response to either antigen is essentially monoclonal, as measured by inhibition of PFC with specific anti-idiotype serum and PC hapten. No age-related alteration in avidity or idiotype expression was observed. Our results demonstrate that no aberrant PC-reactive B cell clones appear in old NZB, and lend support to the notion that the abnormalities observed are due to defective regulatory mechanisms.     

Effect of levamisole on the auto-antibody formation in nude mice.

The effects of levamisole on the formation of antibody to double-stranded DNA (ds-DNA), an auto-antibody, and to some thymus-dependent antigens were studied in nude (nu/nu) mice. The antibody titre to ds-DNA in nu/nu mice treated with levamisole was lower than that in control animals. The number of Thy-1-antigen-positive (Thy-1+) cells in the spleen from nu/nu mice treated with levamisole was higher than that from non-treated animals. It is suggested that the Thy-1+ cells were induced by levamisole in nu/nu mice and these cells suppressed the formation of antibody to ds-DNA, whereas the formation of antibody to a DNP-conjugate (OVA) and SRBC, thymus-dependent antigens, was not evoked in nu/nu mice after the treatment with levamisole.     

B cell immunopoiesis: visualizing the impact of CD40 engagement on the course of T cell-independent immune responses in an Ig transgenic system

This study tracks the fate of antigen-reactive B cells through follicular and extrafollicular responses and addresses the function of CD40 in these processes. The unique feature of this system is the use of transgenic B cells in which the heavy chain locus has been altered by site-directed insertion of a rearranged V(H) DJ(H) exon such that they are able to clonally expand, isotype-switch and follow a normal course of differentiation upon immunization. These Ig transgenic B cells when adoptively transferred into non-transgenic (Tg) mice in measured amounts expanded and differentiated distinctively in response to T cell-independent (TI) or T cell-dependent (TD) antigens. The capacity of these Tg B cells to faithfully recapitulate the humoral immune response to TI and TD antigens provides the means to track clonal B cell behavior in vivo. Challenge with TI antigen in the presence of agonistic anti-CD40 mAb resulted in well-defined alterations of the TI response. In vivo triggering of Tg B cells with TI antigen and CD40 caused an increase in the levels IgG produced and a broadening of the Ig isotype profile, characteristics which partially mimic TD responses. Although some TD characteristics were induced by TI antigen and CD40 triggering, the Tg B cells failed to acquire a germinal center phenotype and failed to generate a memory response. Therefore, TD-like immunity can be only partially reconstituted with CD40 agonists and TI antigens, suggesting that there are additional signals required for germinal center formation and development of memory.