In vitro allotype suppression of spleen cells from immunized rabbits

We tested whether rabbit immune lymphocytes could be suppressed by anti-allotype antibody (Ab) in vitro as shown for normal lymphocytes. Spleen cells (SpC) from rabbits heterozygous at the b locus (b⁴b⁵) of immunoglobulin (Ig) κ chains were treated with IgG preparations of anti-b4 or anti-b5 Ab in vitro for 24 hr (day 1). After this treatment, the SpC were washed and recultured in medium to day 5. The secreted b4- and b5-Ig were quantitated by a radioimmunoassay. SpC from rabbits injected once with sheep red blood cells (SRBC) were allotype-suppressed. Thus, these SpC treated with anti-b4 Ab secreted normal amounts of b5-Ig but secreted much lower amounts of b4-Ig. Similarly, SpC treated with anti-b5 Ab secreted normal amounts of b4-Ig but secreted no detectable b5-Ig. In contrast, SpC from rabbits injected several times with SRBC (hyperimmunized) could not be allotype-suppressed. Hence, the susceptibility of primary immune cells and the resistance of hyperimmune cells to suppression appear to depend on the stage of B-lymphocyte differentiation, presumably because of loss of surface Ig or perhaps because of other changes in the cells as they differentiate during the immune response.     

In vitro Ig heavy chain isotype suppression of spleen cells from immunized rabbits.

We tested whether purified antibodies (Ab) to immunoglobulin (Ig) heavy chain isotoypes could suppress immune Ig-secreting lymphocytes in vitro. Rabbit immune spleen cells (SpC) were treated with purified goat Ab to IgM (anti-μ Ab) or to IgG (anti-γ Ab) in vitro for 24 hr (Day 1). After this treatment, the SpC were washed and recultured to Day 5. The cells were again washed and then tested for Ig-bearing cells by a rosette forming cell assay and tested for Ab-secreting cells by the conventional plaque forming cell assay. In addition, the supernatant fluids were quantitated for secreted Ig by a radial immune hemolysis in gel assay. The number of Ig-bearing cells, the number of Ab-secreting cells and the amount of secreted b4 Ig decreased when “primary immune” SpC were pretreated with anti-μ but not when the SpC were pretreated with anti-γ Ab. Thus, SpC from rabbits injected once with SE were suppressed by anti-μ but not by anti-γ Ab. In contrast, SpC from rabbits injected several times with SE (hyperimmunized) were not suppressed by either anti-μ or by anti-γ Ab. This susceptibility of primary immune (IgM-secreting) SpC and resistance of hyperimmune (IgG-secreting) SpC to suppression may depend on the stage of B lymphocyte differentiation. That is, more differentiated cells such as IgG-secreting cells are insensitive to anti-μ and anti-γ Ab presumably due to lack of surface Ig molecules or for other reasons.     

IL-2 production, IL-2 receptor expression, and IL-2 responsiveness of spleen and mesenteric lymph node cells from inbred mice infected with Trichinella spiralis

The in vitro production of IL-2 and IL-2R expression by lymphoid cells of inbred mice of strong (NFS), intermediate (C3H), or weak (B10.BR) in phenotype of Trichinella spiralis (TS) rejection was measured during a primary infection. Maximum production of IL-2 by spleen and mesenteric lymph node (MLN) cells occurred at 5 days postinfection. Cell depletion experiments demonstrated that Lyt-1.2+ T cells were predominantly responsible for in vitro IL-2 production. Cells from strong-responder NFS mice produced more IL-2 than cells from intermediate-responder C3H or weak-responder B10.BR mice. Similarly, after TS infection, NFS mice had significantly more IL-2R expressing MLN cells than B10.BR or C3H MLN cells. All mouse strains displayed a dose-dependent increase in in vitro IL-2 production after infection with 100 to 800 TS. This effect was most pronounced in NFS mice. Limiting dilution analysis of day 5 infected MLN cells demonstrated that the frequency of TS-reactive CD4+ cells was threefold higher in NFS mice than B10.BR and fourfold higher than in C3H mice. Finally, MLN cells taken from infected NFS mice responded to an exogenous source of IL-2, whereas MLN cells from infected C3H or B10.BR mice were unable to do so. We conclude that strong responsiveness in parasite rejection may be related to the amount of IL-2 produced as well as to the capacity of the lymphocytes of each mouse strain to respond to IL-2. Although these differences help explain the strong rejection phenotype of NFS mice, they fail to separate C3H and B10.BR mice where TS-responsive CD4+ precursors, IL-2 production, and dose responsiveness are all lower for the intermediate phenotype (worm rejection) C3H than the weak phenotype B10.BR mice.     

Modulation of IL-4 production in murine spleen cells by prostaglandins

Recently, it has been reported that IL-4 production by murine Th2 cell lines is insensitive to inhibition by E-type prostaglandins. In the present study, IL-4 production in vitro by freshly isolated concanavalin A (Con A)-stimulated murine spleen cells was readily suppressed by PGE₂ with an I₅₀ of 2 nM. Comparable suppression by PGE₂ was seen after priming by anti-CD3? antibody instead of Con A or with other changes in the culture conditions. PGE₂ was an effective inhibitor after elimination of Ly2.2⁺ T cells, consistent with a direct effect on Th2 cells. In the absence of added prostaglandins, IL-4 production was enhanced 1.5- to 7.0-fold by 0.2–2.0 μM indomethacin, indicating that endogenous arachidonate metabolites such as PGE₂ and PGI₂ regulate IL-4 production in our usual culture system. The inhibition of Th2 cell secretion by PGE₂in vitro may have physiologic and pharmacologic implications for the regulation of Th2 cell function and IgE production in vivo.     

Trypanosoma cruzi-induced suppression of IL-2 production. I. Evidence for the presence of IL-2-producing cells

Mice infected with the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease, are profoundly immunodepressed in their response to various Ag and mitogens. A key factor in this immunosuppression is the essential inability to produce the T cell growth factor IL-2. In this study we demonstrate that this failure to produce IL-2 in response to mitogen stimulation is not the result of the absence of production of soluble or membrane-bound IL-1 by macrophages. Limiting dilution analysis of the precursor frequency of IL-2 producers suggests that an adequate number of precursors for IL-2 production are present in the spleens of infected mice, but that their activity may be regulated by suppressor cells. The presence of precursor cells for IL-2 production is supported by experiments showing that the combination of calcium ionophores and PMA elicits IL-2 production by spleen cells from both normal and T. cruzi-infected mice. Although Con A can provide either of the signals necessary for IL-2 production, calcium flux or protein kinase C activation, to T cells from normal mice, Con A in combination with either calcium ionophore or phorbol ester failed to activate T cells from infected mice to produce IL-2. Preculture of spleen cells from infected mice for 48 to 72 h before addition of Con A results in near normal production of IL-2. This recovery of the capacity to produce IL-2 does not occur if parasite Ag is present during the preculture period. These results suggest that the inability of T cells from T. cruzi-infected mice to produce IL-2 in vitro in response to Con A is not due to the lack of IL-2-producing cells, but may be the result of the maturational state of the T cells or to the presence of a suppressor population.     

Effect of long-term treatment with platelet-activating factor on IL-1 and IL-2 production by rat spleen cells

To study the effect of the in vivo administration of platelet-activating factor (PAF) on cytokine production, alzet minipumps loaded with the mediator or solvent alone were connected to the jugular vein and placed under the skin of Sprague-Dawley rats. Over 7 days the animals received total doses of 0.5, 1, 4.5, 9, or 28 micrograms PAF or the solvent alone. The spleen mononuclear cells isolated from Ficoll gradients and the adherent cell fraction were separated before determination of basal and mitogen-stimulated IL-1 and IL-2 production, respectively. Adherent splenocytes from rats having received 28 micrograms PAF exhibited a decreased capability to produce IL-1, as compared to those from vehicle-treated animals. In contrast, adherent splenocytes from rats having received 9 and 4.5 micrograms PAF yielded higher amounts of released and cell-associated IL-1 activity upon LPS stimulation, as compared to those from solvent-treated animals. The PAF antagonist, BN 52021, given orally (5 mg/kg, twice a day throughout the experiments) inhibited the in vivo effect of 28 micrograms PAF. Statistically significant 144 +/- 43% (p less than 0.001, n = 5) and 73 +/- 33%, (p less than 0.01, n = 3) increases in IL-2 production were observed when whole spleen mononuclear cells from rats administered with 1 and 4.5 micrograms PAF, respectively, were stimulated with Con A. BN 52021 markedly inhibited the in vivo effect of 1 microgram PAF on the IL-2 release. Our study demonstrates that PAF can modulate immune functions in vivo and suggests that the specific PAF antagonist, BN 52021, may be used as an immunomodulatory agent.     

IL-4 production by T cells from naive donors. IL-2 is required for IL-4 production

Utilizing a sensitive and selective assay for IL-4, it was shown that lymph node T cells from naive mice could produce small amounts of this lymphokine in response to anti-CD3 antibodies adsorbed to culture dishes. The capacity of these cells to produce IL-4 in response to plate-bound anti-CD3 was substantially enhanced by the addition of IL-2 to the culture and was strikingly inhibited by monoclonal anti-IL-2 antibody. Thus, IL-2 appears to be essential for IL-4 production by anti-CD3 antibody-stimulated T cells from naive mice. The effect of IL-2 was not mediated either by preferential proliferation or survival of precursors of IL-4 producing cells, indicating that IL-2 regulates T cell production of IL-4. IL-4 producing capacity of T cells from naive mice was found mainly among CD4+ T cells. Large T cells produced much more IL-4, on a per cell basis, than did small T cells. In contrast, small T cells appeared to be equal or superior to large T cells in producing IL-2. The superiority of large T cells in IL-4-producing capacity was not accounted for by a lack of an accessory cell population from the small T cells as addition of large spleen cells depleted of both B and T cells did not enhance IL-4 production by small lymph node T cells. These results suggest that the bulk of IL-4 production by T cell populations, from normal mice, in response to anti-CD3 depends upon cells that are already activated and that IL-2 is required for such production.     

Suppression of interleukin-2 production by human concanavalin A-induced suppressor cells

When PHA-activated normal responder cells (R cells) were cocultured with mononuclear cells (MN cells) which had been preincubated for 48 hr in medium alone (C cells) an enhanced proliferative response was observed. This enhancement was only obtained when the R cells were cultured with allogeneic C cells or when PHA was in the cocultures for the entire culture period. This effect was due to greater production of interleukin 2 (IL-2) by irradiated C cells in the presence of allogeneic or mitogenic stimulation. Con A-treated mononuclear cells (S cells) cultured with PHA-activated allogeneic or autologous responder cells showed reduced [3H]thymidine incorporation and IL-2 production as compared to activated R cells alone. Glutaraldehyde-treated S cells (which retained the ability to absorb IL-2) did not affect the proliferative response or IL-2 production by the R cells, indicating that passive absorption of IL-2 was not entirely responsible for suppression induced by S cells. S cells, pretreated with IL-2, still inhibited R-cell activity. These results show that Con A-treated MN cells suppressed or prevented [3H]thymidine incorporation by actively inhibiting IL-2 production.     

Action of con A-induced suppressor cells on a B hybridoma line

It has been shown that myeloma immunoglobulin production may be regulated by carrier-specific T_H or T_S cells, by idiotype reactive or alloreactive T cells, by antigen itself, or by immune complexes. However, regulation by nonspecific suppressor cells generated by CON A has not hitherto been shown in that system, possibly, it was proposed, due to the committed state of that cell line. We describe here a system in which immunoglobulin production by a hybridoma line is subject to regulation by CON A-activated spleen cells. This system allows us to show clearly that the suppression we observe here is not due to absorption of T_H factor since no such factors are required for antibody production by the hybridoma line. It could still, however, be argued that the suppression observed in these experiments is mediated by macrophages activated by T-cell products. This system has the advantage of being easily dissected to allow further analysis of the mechanism of nonspecific suppression.     

Lipopolysaccharide-induced suppression of airway Th2 responses does not require IL-12 production by dendritic cells

The prevalence of atopic asthma, a Th2-dependent disease, is reaching epidemic proportions partly due to improved hygiene in industrialized countries. There is an inverse correlation between the level of environmental endotoxin exposure and the prevalence of atopic sensitization. As dendritic cells (DC) have been implicated in causing sensitization to inhaled Ag, we studied the effect of endotoxin on Th2 development induced by bone marrow DC in vitro and by intratracheal injection in vivo, with particular emphasis on the role played by the polarizing cytokine IL-12. Bone marrow-derived DC stimulated with Escherichia coli O26:B6 LPS produced IL-12p70 for a limited period of time, after which production became refractory to further stimulation with CD40 ligand, a phenomenon previously called "exhaustion." The level of IL-12 production of DC did not correlate with Th1 development, as exhausted OVA-pulsed DC were still capable of shifting the cytokine pattern of responding OVA-specific Th cells toward Th1 in vitro and in vivo. When mice were first immunized by intratracheal injection of OVA-DC and subsequently challenged with OVA aerosol, prior in vitro stimulation of DC with LPS reduced the development of airway eosinophilia and Th2 cytokine production. Most surprisingly, the capacity of LPS to reduce Th2-dependent eosinophilic airway inflammation was IL-12-independent altogether, as IL-12p40 knockout DC had a similar reduced capacity to prime for Th2 responses. These results suggest that LPS reduces sensitization to inhaled Ag by reducing DC-driven Th2 development, but that IL-12 is not necessary for this effect.     

RU486逆转糖皮质激素对淋巴细胞达白细胞介素—2受体的抑…

本文采用荧光标记ＣＤ２５单抗和放射性配基结合分析实验,观察了ＲＵ４８６地地塞公抑制淋巴细胞表达高、低亲和力ＩＬ－２受本的影响。结果显示,与地塞米松共同培养４８小时的大脾淋细胞,高、低亲和力ＩＬ－２受体的表达明显降低;在含有地塞米松的淋巴细胞培养体系中加入ＲＵ４８６后,表达ＣＤ２５（低亲和力ＩＬ－２受体）的阳性细胞率显著升高,淋巴细胞表面的高亲和力ＩＬ－２受体数量明显增加,基本恢复至正常水平,以上结     

Trypanosoma cruzi-induced suppression of IL-2 production. II. Evidence for a role for suppressor cells

Suppression of IL-2 production during experimental Chagas' disease accounts at least in part for the overall depressed state of the immune system in infected mice. The failure to produce IL-2 in response to mitogen stimulation is not the result of the lack of cells capable of producing IL-2, but appears to be due to regulation of IL-2 production by suppressor cells. This conclusion is supported by cell-mixing experiments where the ability of cells from infected mice to suppress normal spleen cell IL-2 production is evident. Although depletion of plastic and Sephadex G-10 adherent cells results in modest increases in IL-2 production by spleen cells from infected mice, even in the presence of normal adherent cells as a source of IL-1 producers, IL-2 production does not approach normal levels. Also, isolated macrophages are not by themselves suppressive for normal spleen cell IL-2 production, whereas plastic and G-10 nonadherent cells from infected mice are. Depletion of Thy-1+ and Ly-2+ cells not only completely abrogates the ability of spleen cells from infected mice to suppress normal IL-2 production, but results in a cell preparation which actually enhances IL-2 production. Anti-Ly-2 and C treatment of infected spleen cells also markedly enhances their ability to produce IL-2. These results indicate a major role for Ts cells in the regulation of IL-2 production, and a relatively minor role of macrophages as direct effector cells of suppression in this response. The ability to enhance IL-2 production in this system with PG synthesis inhibitors suggests a role for PG-producing cells such as macrophages in the suppressor mechanism, perhaps as inducers of the suppressor effector cells.     

Interferon gamma priming is not critical for IL-12 production of murine spleen cells

Interferon gamma (IFN-gamma) priming is considered to be critical for interleukin 12 (IL-12) production of murine macrophages and human monocytes by lipopolysaccharide (LPS) stimulation. In our present experiments, freshly prepared spleen cells (f-spleen cells) were confirmed not to produce detectable level of IL-12 by LPS stimulation, although they produced significant amount of IL-12 by the stimulation with LPS plus IFN-gamma. However, the stimulation only with LPS induced IL-12 production of spleen cells preincubated in the absence of IFN-gamma. Findings on IL-12 p40 mRNA accumulation were consistent with their IL-12 production. Essentially the same results were obtained using spleen cells from IFN-gamma deficient mice. In the presence of anti-IL-10, f-spleen cells produced IL-12 upon LPS stimulation, indicating that the failure of f-spleen cells in IL-12 production is caused by IL-10 produced by themselves upon LPS stimulation. In addition, f-spleen cells produced IL-12 upon CD40 ligand stimulation, and the production was hardly affected by the presence of IFN-gamma or preincubation. These results indicate that IFN-gamma priming is not critical for IL-12 production of spleen cells stimulated with LPS or CD40 ligand, although IFN-gamma enhances the production, especially, in response to LPS stimulation.     

In vitro immunoglobulin allotype synthesis by spleen cells of heterozygous rabbits. Specific suppression by anti-allotype antibody

The production of immunoglobulin (Ig) bearing the b4 and b5 allotypic markers by b⁴b⁵ heterozygous spleen cells cultured in vitro was assessed by means of a sensitive and reproducible radioimmunoassay. Ig synthesis was demonstrated by the increasing amounts of the b4 and b5 allotypes appearing with time in the supernatant fluids. To determine the effect of anti-b4 or anti-b5 antibody on the synthesis of the b4 and b5 allotypes, spleen cells from b⁴b⁵ heterozygous rabbits were incubated for 24 hr in the presence of anti-b4 or anti-b5 and then washed and cultured for an additional 4 days. Anti-b4 suppressed the production of the b4 allotype with no effect on b5 production, whereas anti-b5 suppressed the production of b5 allotype with no effect on b4 production. This suppression of allotype synthesis in vitro presumably results from an antigen-antibody reaction occurring on the surface of lymphoid cells by a mechanism which may be similar to that which brings about allotype suppression in vivo for fetal and newborn rabbits.     

Selective suppression of IL-12 production by chemoattractants

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1alpha, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-gamma. The production of TNF-alpha and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-beta1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-gamma or by SAC and IFN-gamma, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-gamma production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.     

Lyt-2+ cells. Requirements for concanavalin A-induced proliferation and interleukin 2 production

The requirements for inducing Lyt-2+ T cell proliferation in response to concanavalin A (Con A) were examined. Purified Lyt-2+ or L3T4+ spleen cells of C57BL/6 origin were stimulated with Con A and syngeneic macrophages (MO) in the presence of monoclonal antibodies to T cell markers or to polymorphic determinants on major histocompatibility complex molecules, and assessed for the ability to proliferate and to produce interleukin (IL) 2. alpha I-Ab failed to inhibit the Con A response of Lyt-2+ cells at dilutions that significantly inhibited the response of L3T4+ cells. In contrast, alphaKb/Db or alpha Lyt-2.2 specifically inhibited the response of Lyt-2+ cells, but not L3T4+ cells. The ability of alpha Kb/Db and of alpha Lyt-2.2 to inhibit the response of Lyt-2+ cells was dependent upon the concentration of Con A. These data demonstrate that optimal triggering of T cell subsets to proliferate and to produce IL-2 in response to Con A requires interactions with the appropriate restricting major histocompatibility complex molecule. The role of accessory cells in Lyt-2+ Con A-induced proliferation and IL-2 production was also investigated. Purified Lyt-2+ cells and purified L3T4+ cells failed to respond to Con A in the absence of MO. IL-1 reconstituted the response when MO were limiting, but failed to restore the response of either Lyt-2+ or L3T4+ cells when T cells were rigorously purified to remove all MO. These results demonstrate that triggering Lyt-2+ T cells, like L3T4+ T cells, requires accessory cells, and that this does not merely reflect a requirement for IL-1 production. Thus, Con A-induced proliferation and IL-2 production by Lyt-2+ T cells requires intimate contact with accessory cells and interactions dependent upon the class I-restricting element.     

Production of IL-13 in spleen cells by IL-18 and IL-12 through generation of NK-like cells

Treatment of Nylon wool-passed cells (NWC) prepared from the spleen of C57BL/6 mice with IL-18 and IL-12, but not with IL-18 alone, resulted in induction of IFN-gamma, a Th1 cytokine, and GM-CSF at 24 h, and IL-13, a Th2 cytokine at 72 h. The induction of IL-13 was suppressed by anti-GM-CSF antibody, indicating involvement of GM-CSF in IL-13 production. When NWC incubated with IL-18 and IL-12 for 72 h ("primary treatment") were treated again with the same cytokines ("secondary treatment"), IL-13 was induced much more quickly than observed in the primary treatment. Flow cytometric analysis of NWC after the primary treatment showed marked increases in the CD4(-)CD8(-) non-T cell population bearing CD25(+), CD45RB(super high) and CD122(+). These cells were positive for CD49b but negative for NK1.1, indicating that they were not typical but NK-like cells. The NK-like cells produced IL-13 in response to the treatment with IL-18 alone, indicating that the generation of these cells in the primary treatment likely accounts for the quick production of IL-13 in the secondary treatment. These results show that IL-18 and IL-12 generates the NK-like cells in NWC by a process mediated by GM-CSF that are ready for producing IL-13.     

The ability of peripheral blood mononuclear cells (PBMC) of syphilitic patients to produce IL-2

Abstract The cell-mediated immune response of importance in protection against Treponema pallidum , is distinctly suppressed in some stages of the disease. This may be a result of decreased ability of cells to produce IL-2, or IL-2 absorption by different factors. The experiments were designed to evaluate the ability of peripheral blood mononuclear cells (PBMC) of patients with different stages of syphilis to produce IL-2, and to investigate the causes which could possibly limit its activity. The ability of the PBMC of syphilitic patients to produce IL-2 develops at the beginning of the disease, reaching a maximum in primary seropositive syphilis. In the next stages of the disease this capability is distinctly lowered. The lowest was in malignant syphilis and tabes dorsalis, i.e. during severe disease. Absorption of adherent cells from PBMC increased the ability of lymphocytes to produce IL-2. The highest level of this interleukin was observed at the stages of the disease where suppression was the deepest. Sera of both control and syphilitic patients contained IL-2 inhibitor. Its level was the highest in early and late latent syphilis where no symptoms of disease were present. In all syphilitic sera a distinctly elevated level of soluble IL-2 receptors (sIL-2R) was also found. Its high level was noted in sera of patients in which PBMC had the weakest ability to produce IL-2. These findings suggest that sIL-2R may be bound to IL-2 and in this way would lead to weakening of T cell function and of resistance against Treponema pallidum infection.