Interleukin-8 differentially modulates interleukin-4- and interleukin-2-induced human B cell growth

The effect of interleukin-8 (IL)-8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL-8 inhibited IL-4-induced growth of B cells costimulated with anti-mu antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose-dependent fashion. In contrast, IL-8 did not inhibit IL-2-induced growth of B cells. The IL-8-mediated inhibition was specific, since it was blocked by anti-IL-8 mAb but not by control IgG1. Moreover, anti-tumor necrosis factor-alpha (anti-TNF-alpha) Ab blocked IL-8-mediated inhibition. On the other hand, TNF-alpha, but not other cytokines including IL-1 beta, IL-3, IL-5, IL-6, interferon-alpha (IFN-alpha) or IFN-gamma, inhibited IL-4-mediated growth, and inhibition by TNF-alpha was blocked by anti-TNF-alpha Ab but not by control IgG. IL-4 had no effect on TNF-alpha binding by B cells while it decreased TNF-alpha production by B cells. IL-8 had no effect in binding of IL-4, IL-2 or TNF-alpha by B cells, however, it enhanced TNF-alpha production by B cells. These results indicate that IL-8 inhibited IL-4-induced human B cell growth by enhancement of endogenous TNF-alpha production.     

Regulation of surface and intracellular expression of CTLA4 on mouse T cells

CTLA4 is a cell surface molecule that shares 30% homology with CD28 and binds B7 family members with high affinity. Analysis of surface expression on murine T cells revealed up-regulation after stimulation with anti-CD3 mAb in vitro and further augmentation after the addition of exogenous IL-2 or anti-CD28 mAb. The effects of IL-2 and anti-CD28 mAb were additive and in part independent, as anti-CD28 mAb increased anti-CD3 mAb-induced T cell CTLA4 expression in IL-2-deficient mice. In contrast, CTLA4 expression was only minimally augmented by the addition of IL-4, IL-6, IL-7, or IL-12. Expression of CTLA4 induced by anti-CD3 mAb was inhibited by anti-IL-2 plus anti-IL-2R mAbs. Inasmuch as these agents prevented T cell proliferation, the effects of cell cycle inhibitors also were examined. Drugs blocking at G1 (cyclosporin A, mimosine) or S (hydroxyurea) phase inhibited the up-regulation of CTLA4 induced by anti-CD3 mAb, suggesting that entry into the cell cycle was necessary to increase the expression of CTLA4. The kinetics of intracellular expression of CTLA4 after stimulation with anti-CD3 mAb paralleled those of surface expression, but surprisingly, much more CTLA4 was localized in the cytoplasm of T lymphocytes than on the cell surface at each time point. Importantly, surface CTLA4 was rapidly internalized intracellularly, which may explain the low levels of expression generally detected on the cell surface. We conclude that both CD28 and IL-2 play important roles in the up-regulation of CTLA4 expression. In addition, the cell surface accumulation of CTL4 appears to be primarily regulated by its rapid endocytosis.     

Establishment of a novel myeloma cell line KPMM2 carrying t(3;14)(q21;q32), which proliferates specifically in response to interleukin-6 through an autocrine mechanism

We established a new human myeloma cell line, KPMM2, which proliferates specifically in response to IL-6 via an autocrine mechanism. The proliferative response of KPMM2 cells to exogenous IL-6 was significantly stimulated in a dose-dependent manner. The growth was markedly inhibited by an anti-IL-6 mAb and an anti-IL-6 receptor (IL-6R) mAb in a dose-dependent manner. KPMM2 cells expressed IL-6 and IL-6R mRNA by RT-PCR. Flow cytometric analysis showed cell surface expression of IL-6R. IL-6 protein was detected in the culture supernatant by ELISA. IL-11, oncostatin M and leukemia inhibitory factor had no effect on the proliferation of KPMM2 cells although interferon-alpha and interferon-gamma inhibited the growth. Furthermore, KPMM2 cells bore a t(3;14)(q21;q32) translocation and this finding is of potential interest for future studies in the light of the nuclear protein BM28 (CDCL1, for cdc-like 1) mapped on 3q21, which plays an important role in the cell cycle. In this report, we demonstrated completely an IL-6-dependent autocrine growth mechanism in KPMM2 cell line. This cell line may be useful to investigate the pathogenesis of multiple myeloma and to evaluate the therapeutic potential of IL-6 blocking agents in vitro and in vivo.     

Monomeric IgG2 enhances Ig production and proliferation in human B cells

The effect of purified polyclonal human IgG subclasses on B-cell responses was studied using the human IgA-producing B-cell line GM-1056. IgG2 at concentrations of 0.01-1 microgram/mL enhanced both IgA production and proliferation, while IgG1, IgG3, and IgG4 each failed to do so at tested concentrations between 0.001 and 10 micrograms/mL. This enhancement was Fc gamma R mediated, since IgG2 Fc fragments enhanced IgA production and proliferation to the same extent as did the whole IgG2 molecule, whereas F(ab')2 fragments did not. However, in contrast to monomeric IgG2, aggregated IgG2, which was expected to bind Fc gamma RII on B cells, affected neither IgA production nor proliferation. Similarly, anti-CDw32 mAb (2E1, anti-Fc gamma RII), anti-CD 64 mAb (32.2 anti-Fc gamma RI), and anti-CD16 mAb (Leu 11a, anti-Fc gamma RIII) mAb each failed to stimulate GM-1056 cells, and more importantly did not block IgG2-induced stimulation. Of various cytokines tested, including IFN-alpha, IFN-gamma, IL-1 beta, IL-2, IL-3, IL-4, IL-5, and IL-6, IL-6 alone augmented IgG2-induced enhancement of IgA production and proliferation. Moreover, the IL-6 effect was lost following preabsorption with anti-IL-6 antibody but not following preabsorption with control antibody. IgG2 also enhanced Ig production and proliferation in tonsillar large activated B cells, while IgG1, IgG3 and IgG4 each failed to do so. In contrast, IgG2 had no effect on Ig production and proliferation in tonsillar small resting B cells or SAC-stimulated small B cells. IgG2-induced enhancement of Ig production and proliferation in large B cells was not blocked by 2E1, 32.2, or Leu 11a, while enhancement was augmented in a specific fashion by IL-6. These results indicate that monomeric IgG2 specifically enhances B cell responses via an Fc gamma R receptor distinct from Fc gamma RI, Fc gamma RII, and Fc gamma RIII, and that IL-6 may play a role in augmenting this response.     

Anti-CD3-activated killer T cells: Interleukin-6 modulates the induction of major histocompatibility complex-unrestricted cytotoxicity and the expression of genes coding for cytotoxic effector molecules

We have investigated the role of interleukin-6 (IL-6) in the induction of major histocompatibility complex (MHC)-unrestricted cytotoxicity, as well as granzyme B, perforin, and Fas ligand gene expression, following mouse T lymphocyte activation with anti-CD3 monoclonal antibody (mAb). The generation of anti-CD3-activated killer-T (AK-T) cells was inhibited when anti-IL-6 neutralizing mAb was added at initiation of culture but not 24 h later, indicating that IL-6 is involved at an early stage of AK-T cell development. However, AK-T cell induction in the presence of exogenous IL-6 did not result in enhanced cytotoxicity, suggesting that saturating levels of IL-6 are normally synthesized in AK-T cell cultures. The inhibitory effect of IL-6 neutralization on AK-T cell generation could not be attributed to a defect in AK-T cell proliferation or to an inability of AK-T cells to recognize and adhere to P815 tumor target cells. However, IL-2 synthesis and CD25 expression were downregulated in AK-T cell cultures performed in the presence of anti-IL-6 mAb. In addition, IL-6 neutralization resulted in decreased expression of granzyme B and perforin, but not Fas ligand, mRNA. Exogenous IL-2 (50 U/ml) added at initiation of culture completely reversed the inhibitory effect of anti-IL-6 mAb on AK-T cell development, restoring CD25 expression and tumoricidal activity, as well as granzyme B and perforin mRNA expression, to control levels. We conclude that IL-6 modulates AK-T cell induction through an IL-2-dependent mechanism.     

Recombinant soluble murine IL-4 receptor can inhibit or enhance IgE responses in vivo

This study examines the effects of soluble IL-4R (sIL-4R) administration on IgE production in vivo by using an anti-IgD injection model. Anti-IgD-treated mice were given various doses of sIL-4R or anti-IL-4 mAb over a 3-day period and serum IgE levels were determined by ELISA on day 9. The sIL-4R inhibited IgE production by up to 85%. Anti-IL-4 mAb administration resulted in comparable levels of inhibition at considerably lower doses. The disparity in efficacy between sIL-4R and anti-IL-4 mAb was likely the result of differences in the biodistribution and in vivo half-life of the two IL-4-binding proteins. The specificity of the sIL-4R inhibitory effect was assessed by mixing sIL-4R with various concentrations of IL-4 before injection. Exogenous IL-4 partially overcame the inhibitory effect of high-dose sIL-4R or anti-IL-4 mAb. Unexpectedly, coadministration of suboptimal concentrations of anti-IL-4 mAb or sIL-4R with IL-4 resulted in superinduction of the IgE response. This stimulatory effect was dose dependent for both IL-4 and the IL-4 cognates and was not seen in the absence of exogenous IL-4 over the entire concentration range tested for either sIL-4R or anti-IL mAb. The results indicate that sIL-4R can block IgE secretion by neutralizing endogenous IL-4. Furthermore, sIL-4R can enhance, in a dose-dependent manner, the biologic effects of exogenously administered IL-4, presumably by altering the biodistribution of the cytokine. These findings suggest two alternative applications for cytokine-binding proteins, i.e., 1) as antagonists of biologic activities of endogenously produced cytokines and, 2) as vehicles for cytokine delivery.     

Mechanisms of multiple myeloma cell growth

The mechanism of human multiple myeloma cell growth was studied utilizing eleven myeloma cell lines established in vitro or in vivo (Scid mouse). Four bone marrow derived cell lines grew dependently on IL-6 or bone marrow stromal cells. Seven extramedullary lesion derived cell lines grew spontaneously and additively proliferated in response to IL-6. All cell lines expressed the IL-6 receptor (IL-6R) and IL-6RmRNA, but none expressed IL-6mRNA. No IL-6 activity was detected in the myeloma cell culture supernatant. Both the anti-IL-6 antibody and anti-IL-6R antibody neutralized IL-6-induced proliferation, but did not inhibit spontaneous proliferation of extramedullary lesion derived cell lines. While establishing cell lines, it was found that the proliferating fraction was primarily included in a fraction which was non-adherent to stromal cells and composed of undifferentiated plasmablasts. Undifferentiated plasmablasts proliferated in response to IL-6, in contrast to the adherent, mature form of myeloma cells which did not proliferate in response to IL-6. Innoculation of myeloma cells into Scid mice induced subcutaneous tumor formation. These tumors were composed of undifferentiated plasmablasts, which also proliferated in response to IL-6. These results imply that the growth of bone marrow derived myeloma cell lines are dependent on the IL-6 paracrine mechanism and that the growth of extramedullary lesion derived cell lines primarily autonomous and additively dependent on the IL-6 paracrine mechanism.     

Identification of an IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). I. Production of the non-IL-7 component by bone marrow stromal cells from IL-7 gene-deleted mice

Mouse bone marrow (BM) stromal cell conditioned medium (CM) from our long-term lymphoid culture system selectively induces the in vitro proliferation and presumptive differentiation of pre-pro-B cells (B220+, HSA-, TdT- or TdT+, c[mu-]) from adult rat, mouse, and human BM. However, the responsible growth factor(s) has not yet been identified. Inasmuch as IL-7 is one of the cytokines most closely associated with early B-lineage development, we utilized BM adherent cells and stromal cell lines from IL-7 gene-deleted (-/-) mice in combination with rIL-7 and anti-IL-7 mAb to investigate its possible regulatory role in our culture system. The results show that, although rIL-7 and IL-7 (-/-) CM each can maintain the viability of freshly harvested pre-pro-B cells in vitro, neither induces them to proliferate and/or differentiate, even in the presence of recombinant stem cell factor (rSCF) and/or recombinant insulin-like growth factor (rIGF). The results also show that anti-IL-7 mAb fails to neutralize the pre-pro-B cell growth-stimulating activity in IL-7 (+/+) CM. Yet rIL-7 enables IL-7 (-/-) CM to induce proliferation of pre-pro-B cells, and to "prime" them to respond directly to monomeric IL-7. Furthermore, anti-IL-7 mAb adsorbs the pre-pro-B cell growth-stimulating activity from both IL-7 (+/+) CM and rIL-7-supplemented IL-7 (-/-) CM; but rIL-7 does not restore this activity. Lastly, both pre-pro-B cell growth-stimulatory activity and IL-7 are quantitatively recovered by ultrafiltration in the 50 to 100 kDa, rather than the 10 to 50 kDa, apparent molecular mass fraction. These results suggest that the pre-pro-B cell growth-stimulating activity in our culture system is the property of a self-associating complex of IL-7 and a second BM stromal cell-derived cofactor.     

In vitro interleukin 12 activation of peripheral blood CD3(+)CD56(+) and CD3(+)CD56(-) gammadelta T cells from glioblastoma patients

Interleukin (IL)-12 has recently been shown to be directly involved in the activation of natural killer and alphabeta T cells via an IL-2-independent pathway. We show here that another type of human cytotoxic cell, gammadelta T cells activated by solid-phase anti-CD3 antibody and expanded using IL-2, obtained, in this case, from the peripheral blood of glioblastoma patients, displays significant tumoricidal activity. In addition, its cytotoxic activity against K562 or Daudi cells or against autologous glioblastoma targets (but not lymphocytes) is significantly enhanced when costimulated with IL-2 and IL-12. To study this synergistic activation by the two interleukins of the patients' gammadelta T cells, we screened the cells for the presence of the IL-2 receptor (IL-2R) and IL-12 receptor (IL-12R) using both flow cytometric analysis and PCR. The patients' gammadelta T cells constitutively expressed the high-affinity IL-2R; when stimulated with IL-12 plus IL-2, the levels of IL-2Ralpha and IL-2Rbeta increased, whereas that of IL-2gamma did not. They also expressed marginal levels of low-affinity IL-12R both immediately after IL-2 expansion and after 24-h incubation, and significantly higher levels after 72-h incubation, consistent with the level of gammadelta T-cell activation. IL-12 alone induced little proliferation of patients' gammadelta T cells in a 24-h assay and none in a 72-h assay; however, it caused a marked inhibition of the IL-2-induced proliferative response in the 72-h assay. The synergistic action of IL-2 and IL-12 was completely abolished by combined pretreatment with anti-IL-2alpha, beta, and gamma mAbs. IL-12-mediated enhancement of gammadelta T cell cytotoxic activity was inhibited by anti-IL-2Rbeta mAb in a dose-dependent manner but not by anti-IL-2Ralpha or anti-IL-2Rgamma mAbs. Thus, the increased expression of the IL-2Rbeta is critical for the synergistic activation of gammadelta T cells by IL-12 plus IL-2; it is also probable that at least the low-affinity IL-12R contributes to the activation of gammadelta T cells mediated by either IL-12 alone or IL-12 plus IL-2. We have, therefore, demonstrated that IL-12 can stimulate the cytotoxic activity of gammadelta T cells from glioblastoma patients, acting via the IL-2Rbeta component of the IL-2R and low-affinity IL-12R. IL-12 activation of patients' gammadelta T cells could possibly be of potential use in the treatment of glioblastoma patients.     

Interleukin 13 inhibits growth of human renal cell carcinoma cells independently of the p140 interleukin 4 receptor chain

Interleukin-13 (IL-13) is a cytokine produced primarily by activated T lymphocytes. It exerts a variety of effects on different cell types, including monocytes, B lymphocytes, mast cells, and keratinocytes. The effects of IL-13 on target cells are often similar to the effects of IL-4, which is another cytokine product of activated T lymphocytes. We recently described the expression of intermediate- to high-affinity receptors for IL-13 (IL-13R) on renal cell carcinoma (RCC) cells. In the present study, we examined the effect of IL-13 on the growth of RCC cells as measured by [3H]thymidine uptake and a clonogenic assay. In addition, we used an IL-4R-specific antibody to examine the specificity of IL-4R and IL-13R binding and function. We observed that IL-13 inhibited RCC cell proliferation by up to 50% and colony formation by up to 32% when compared with cells cultured in medium alone. A combination of IL-4 and IL-13 did not have an additive or synergistic effect on the growth of RCC cells. These cells expressed mRNA for IL-13 and secreted immunoreactive IL-13 protein in culture. The growth-inhibitory effects of IL-13 were specific, because they were not affected by antibodies to IL-4 or to the 140-kilodalton subunit of IL-4R. Furthermore, polyclonal antibodies to IL-4R failed to inhibit the binding of 125I-IL-13 to RCC cells. These results indicate that IL-13 has significant antiproliferative effects on human RCC cells, and the inhibition of IL-13 effects by anti-IL-4R antibody previously reported in lymphoid cells does not occur in RCC cells.     

Immortalization with herpesvirus saimiri modulates the cytokine secretion profile of established Th1 and Th2 human T cell clones

T blasts of six established human CD4+ T cell clones with defined Ag specificity and cytokine secretion profile (3 Th1 and 3 Th2) were immortalized with Herpesvirus saimiri (HVS) and compared with their uninfected counterparts for their ability to proliferate, produce cytokines, and express cytolytic activity. HVS-transformed Th1 and Th2 clones neither substantially changed their original surface markers nor lose their ability to proliferate in response to their specific Ag but did acquire the ability to proliferate in response to contact signals delivered by SRBC or autologous APC alone. In addition, transformation by HVS substantially enhanced the lectin-dependent cytolytic activity of Th1 clones and enabled noncytolytic Th2 clones to exert cytolytic activity. HVS-transformed Th1 clones but not their uninfected counterparts spontaneously transcribed and secreted Th1-type cytokines (IL-2, IFN-gamma, and TNF-beta) and such a production was further enhanced by stimulation with either SRBC or PMA plus anti-CD3 mAb. HVS transformed but not uninfected Th2 clones constitutively expressed both IL-4 and IL-2 mRNA and secreted IFN-gamma. Stimulation with PMA plus anti-CD3 mAb induced uninfected Th2 clones to secrete high amounts of IL-4 and IL-5 but not Th1-type cytokines, whereas the same HVS-transformed Th2 showed minimal IL-4 and IL-5 secretion with concomitant high production of IL-2, IFN-gamma, and TNF-beta. Transformation by HVS also resulted in up-regulation of TNF-alpha and IL-3 production by both Th1 and Th2 clones. The ongoing proliferation of HVS-transformed clones was partially inhibited by either anti-IL-2 or anti-IL-3 antibodies and virtually abolished by the combined addition of the two anticytokine antibodies, suggesting that both IL-2 and IL-3 can function as autocrine growth factors for HVS-transformed Th1 and Th2 clones.     

Cuticular proteins from the lobster, Homarus americanus

The interleukin-12 receptor (IL-12R)beta1 chain is an essential component of the functional IL-12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti-CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up-regulation of IL-12Rbeta1 expression and IL-12 binding. Kinetic studies revealed that maximum expression of IL-12Rbeta1 and IL-12 binding occurred on days 3-4. Anti-CD3-induced expression of IL-12Rbeta1 chain and IL-12 binding by PBMC was augmented by anti-CD28 mAb, indicating that the potentiating effect of anti-CD28 on T cell responses to IL-12 could be mediated, at least in part, by the enhancement of IL-12R expression. Among 16 cytokines tested, IL-2, IL-7 and IL-15 markedly induced IL-12Rbeta1 expression and IL-12 binding on resting PBMC, whereas IL-1alpha and tumor necrosis factor-alpha had a minimal enhancing effect. In contrast, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-alpha, IFN-gamma, granulocyte/macrophage colony-stimulating factor and transforming growth factor (TGF)-beta2 had no detectable enhancing effect. Anti-CD3-induced expression of IL-12Rbeta1 and of low-affinity IL-12 binding sites was partially inhibited by TGF-beta2, IL-10 and IL-4; however, TGF-beta2 and IL-10 completely abolished anti-CD3-induced expression of high-affinity IL-12 binding sites. Consistent with the reduction of high affinity IL-12 binding sites, PBMC activated with anti-CD3 mAb in the presence of TGF-beta2 or IL-10 failed to produce IFN-gamma or to proliferate in response to IL-12. These results suggest that Th2 cell-derived cytokines can inhibit IL-12-induced biological functions by inhibiting IL-12R expression and that expression of a second subunit of the IL-12R (IL-12Rbeta2), required for the formation of high-affinity IL-12 binding sites, may be more highly regulated by TGF-beta2 and IL-10 than is expression of IL-12Rbeta1.     

Human naive CD4 T cells produce interleukin-4 at priming and acquire a Th2 phenotype upon repetitive stimulations in neutral conditions

The maturation of naive CD4 T cells into interleukin (IL)-4-producing effectors was shown to require the presence of IL-4 at priming, the cellular origin of which remains unclear. We demonstrate here that naive T cells themselves release IL-4 at very low levels that are nevertheless sufficient to promote their development into Th2-like cells. This conclusion is based on three observations: (1) highly purified human naive CD4 T cells, of neonatal or adult origin, develop into Th2 effectors upon repetitive cycles of stimulation with anti-CD3 monoclonal antibody (mAb) cross-linked to CD32-B7 transfected L fibroblasts followed by IL-2 expansion; (2) IL-4 protein is readily detectable in the concentrated supernatant fluids of priming cultures performed in the presence of anti-IL-4 receptor mAb; and (3) addition of anti-IL-4 or anti-IL-4 receptor mAb at priming markedly inhibits the acquisition of IL-4- and IL-5-producing capacity while enhancing that of interferon-gamma.     

Human T cells require IL-2 but not G1/S transition to acquire susceptibility to Fas-mediated apoptosis

The interaction between Fas ligand and Fas, both expressed on activated T cells, is the major pathway in the regulation of activation-induced cell death. However, activated T cells that express membrane Fas are initially resistant to anti-Fas-induced apoptosis and become susceptible only after proliferation in vitro. Since IL-2 is known to regulate activation-induced cell death, we studied the effect of IL-2 on anti-Fas-mediated apoptosis. Interference with the IL-2 pathway was achieved by 1) inhibition of cytokine synthesis using cyclosporin A or FK506, 2) neutralization of IL-2 by anti-IL-2 Ab, 3) inhibition of binding to IL-2R by CD25 mAb, and 4) blocking of IL-2R signaling by rapamycin. We show that Fas expression is independent of the IL-2 pathway, whereas Fas-mediated apoptosis does not develop in the presence of inhibitors of IL-2 production or signaling. While the addition of rIL-2 reversed the inhibitory effect of cyclosporin A and FK506, the addition of rIL-4, rIL-7, or rIFN-gamma did not, although these cytokines induced progression into the S phase of the cell cycle. Aphidicolin-treated activated T cells that do not progress into the S phase were susceptible to Fas-mediated apoptosis. Therefore, Fas-mediated apoptosis is controlled by signals generated by IL-2 in agreement with the reported alteration of apoptosis in mice deficient in IL-2 or IL-2R.     

Induction of tolerance with nondepleting anti-CD4 monoclonal antibodies is associated with down-regulation of TH2 cytokines

BACKGROUND: Induction of tolerance with anti-CD4 has mainly focused on monoclonal antibodies (mAbs) that deplete CD4+ T cells. In this study, the mechanisms by which nondepleting anti-CD4 mAbs induce tolerance in the Dark Agouti to PVG rat heart graft model were examined. METHODS: Five anti-CD4 mAbs were tested. Immunohistology and cytokine mRNA profiles were analyzed within grafts. Effects of combining anti-CD4 therapy with alloantibody (alloAb), interleukin (IL)-4, and anti-IL-4 mAb were also examined. RESULTS: All mAbs tested induced indefinite graft survival (>150 days), with blocking of alloAb production. Exogenous alloAb did not restore rejection. Similar T cell receptor alphabeta+, CD8+, IL-2 receptor+ T cell, macrophage, and natural killer cell infiltration and comparable MHC II and intercellular adhesion molecule-1 levels were seen in rejecting and tolerant grafts. mRNA for IL-2, interferon-gamma, lymphotoxin, tumor necrosis factor-alpha, transforming growth factor-beta, cytolysin, and granzyme-A/B was comparable, although inducible nitric oxide synthase was slightly reduced in tolerant grafts. IL-4 and IL-5 were significantly reduced in tolerant grafts, although IL-6, IL-10, and IL-13 levels were similar; this was consistent with partial T helper (Th)2 response inhibition, which was also manifested by inhibited alloAb. The combination of alloAb, IL-4, or anti-IL-4 mAb with anti-CD4 did not prevent tolerance induction. CONCLUSIONS: This study demonstrated that anti-CD4 mAb therapy did not inhibit activation and infiltration of Th1 and CD8+ effector T cells. Preferential induction of Th2 responses, especially IL-4, was not essential for the induction of tolerance. Our studies also found no evidence to support induction of anergy or transforming growth factor-beta as mechanisms of tolerance induction. These results question whether IL-4 is required for induction of transplantation tolerance.     

Leishmania major-infected C3H mice treated with anti-IL-12 mAb develop but do not maintain a Th2 response

Leishmania major-infected C3H mice develop a Th1 response, but studies have shown that treatment of C3H mice with anti-IL-12 or anti-IFN-gamma mAb promotes the development of a Th2 response and susceptibility. However, we discovered that C3H mice treated for 3 wk with either anti-IL-12 or anti-IFN-gamma mAb eventually resolved their lesions and switched from a Th2 to a Th1 response. No significant differences in IL-4, IL-10, or IFN-gamma levels or in the parasite burden could be detected between BALB/c and anti-IL-12-treated C3H mice early after infection, suggesting that the instability of the Th2 response in anti-IL-12-treated C3H mice was unrelated to levels of these cytokines and parasite numbers. However, anti-IL-12-treated C3H mice continued to produce IL-12 in spite of exhibiting a Th2 phenotype. To determine whether the production of IL-12 was associated with the healing observed in these animals, we treated C3H mice with anti-IL-12 continuously for 12 wk. In contrast to C3H mice given anti-IL-12 for 3 wk, C3H mice continuously treated with anti-IL-12 failed to heal. These results suggest that qualitative differences in Th2-type responses may influence their stability and that the presence of IL-4, IL-10, or high parasite numbers is not sufficient to maintain a Th2 response in mice with certain genetic backgrounds.     

Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice

We investigated the mechanisms by which treatment with anti-IL-12 Ab prevents cure of infection with Leishmania major in resistant C57BL/6 mice. Consistent with delayed production of IL-12, anti-IL-12 Abs could be administered as late as 2 wk after infection to exacerbate disease. Starting at 2 wk of infection, the cultured lymph node cells from mice treated with either polyclonal or monoclonal anti-IL-12 Abs persistently generated 3- to 10-fold more IL-4 and IL-10 in response to L. major Ag compared with cells from mice receiving preimmune goat IgG. Reciprocal decreases in Ag-specific IFN-gamma production were observed in mice receiving anti-IL-12 Abs. A similar reversal of IFN-gamma and IL-4 production accompanied progressive disease induced by pretreatment with a single dose of anti-IFN-gamma mAb. Although IFN-gamma production was suppressed for up to 4 wk in mice treated with monoclonal anti-IL-12 or anti-IFN-gamma, coadministration of neutralizing anti-IL-4 IgG reversed progressive illness. These findings demonstrate that IL-12 produced in vivo is necessary for both the emergence of IFN-gamma producing cells and the down-regulation of Th2 cell responses during murine leishmaniasis. Furthermore, the uninhibited production of IL-4 was required to sustain progressive infection initiated by the decreased IFN-gamma synthesis observed in anti-IL-12 and anti-IFN-gamma-treated mice.     

IL-12, but not IFN-gamma, plays a major role in sustaining the chronic phase of colitis in IL-10-deficient mice

IL-10-deficient (IL-10(-/-)) mice develop chronic enterocolitis mediated by CD4+ Th1 cells producing IFN-gamma. Because IL-12 can promote Th1 development and IFN-gamma production, the ability of neutralizing anti-IL-12 mAb to modulate colitis in IL-10(-/-) mice was investigated. Anti-IL-12 mAb treatment completely prevented disease development in young IL-10(-/-) mice. Treatment of adult mice resulted in significant amelioration of established disease accompanied by reduced numbers of mesenteric lymph node and colonic CD4+ T cells and of mesenteric lymph node T cells spontaneously producing IFN-gamma. In contrast, anti-IFN-gamma mAb had minimal effect on disease reversal, despite a significant preventative effect in young mice. These findings suggested that IL-12 sustains colitis by supporting the expansion of differentiated Th1 cells that mediate disease independently of their IFN-gamma production. This conclusion was supported by the finding that anti-IL-12 mAb greatly diminished the ability of a limited number of CD4+ T cells expressing high levels of CD45RB from diseased IL-10(-/-) mice to expand and cause colitis in recombination-activating gene-2(-/-) recipients, while anti-IFN-gamma mAb had no effect. Furthermore, IL-12 could support pathogenic IL-10(-/-) T cells stimulated in vitro in the absence of IL-2. While these studies show that IL-12 plays an important role in sustaining activated Th1 cells during the chronic phase of disease, the inability of anti-IL-12 mAb to abolish established colitis or completely prevent disease transfer by Thl cells suggests that additional factors contribute to disease maintenance.