Regulation of interleukin-12 receptor beta1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells.

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.

Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice.

Collagen-induced arthritis (CIA) is an animal model for rheumatoid arthritis. The disease is elicited by immunization of genetically susceptible DBA/1 mice with type II collagen, resulting in a debilitating arthritis characterized by inflammation and involvement of multiple joints. We investigated the role of endogenous interleukin (IL)-12 in the pathogenesis of this disease by undertaking an analysis of IL-12-deficient mice on the DBA/1 genetic background after immunization with type II collagen. Both the incidence and severity of disease were significantly reduced in mice unable to produce biologically active IL-12. Concomitant decreases were observed in serum levels of pathogenic, collagen-specific IgG2a antibodies and collagen-induced secretion of interferon-gamma by immune splenocytes in vitro, consistent with an impaired T helper-1 response. There were, however, a few animals which developed severe disease in a single paw in spite of this highly diminished Th1 response. Taken together, these results demonstrate an important role for IL-12 in the pathogenesis of CIA, although it is not absolutely required for disease development.

Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo.

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis.

T cell cytokines are known to play a major role in determining protection and pathology in infectious disease. It has recently become clear that IL-12 is a key inducer of the type 1 T cell cytokine pattern characterized by production of IFN-gamma. Conversely, IL-10 down-regulates IL-12 production and type 1 cytokine responses. We have investigated whether IL-12 and IL-10 might be involved in a chronic inflammatory reaction, atherosclerosis. In atherosclerotic plaques, we found strong expression of IFN-gamma but not IL-4 mRNAs as compared to normal arteries. IL-12 p40 mRNA and IL-12 p70 protein were also found to be abundant in atherosclerotic plaques. IL-12 was induced in monocytes in vitro in response to highly oxidized LDL but not minimally modified LDL. The cross-regulatory role of IL-10 was indicated by the expression of IL-10 in some atherosclerotic lesions, and the demonstration that exogenous rIL-10 inhibited LDL-induced IL-12 release. These data suggest that the balance between IL-12 and IL-10 production contributes to the level of immune-mediated tissue injury in atherosclerotsis.

IL-12-deficient mice are defective in IFN gamma production and type 1 cytokine responses.

IL-12 is a cytokine that can exert regulatory effects on T and NK cells and promote Th1 responses. To delineate further the physiologic role of IL-12 in immunity, mice deficient for this cytokine were generated. IL-12-deficient mice were impaired but not completely lacking in the ability to produce IFN gamma following endotoxin administration and to mount a Th1 response in vivo, as measured by antigen-induced IFN gamma secretion by immune lymph node cells in vitro. In contrast, secretion of IL-4 was enhanced, while proliferation and secretion of IL-2 and IL-10 were normal following antigen stimulation. DTH responses were significantly reduced in IL-12-deficient mice, but no defect in allogeneic CTL responses was observed. These results indicate that IL-12 plays an essential role in regulating IFN gamma production and in facilitating normal DTH responses. However, other phenomena associated with Th1 responses and cell-mediated immunity, i.e., IL-2 secretion and CTL generation, were not compromised in the absence of IL-12.

Biological function and distribution of human interleukin-12 receptor beta chain.

We previously described the cloning of a cDNA encoding an interleukin-12 receptor (IL-12R) subunit, designated beta, that bound IL-12 with low affinity when expressed in COS cells. We now report that a pair of monoclonal antibodies (mAb), 2B10 and 2.4E6, directed against different epitopes on the IL-12R beta chain, when used in combination, strongly inhibited IL-12-induced proliferation of activated T cells, IL-12-induced secretion of interferon-gamma by resting peripheral blood mononuclear cells (PBMC), and IL-12-mediated lymphokine-activated killer cell activation. The mAb had no effect on lymphoblast proliferation induced by IL-2, -4, or -7. Thus, the IL-12R beta chain appears to be an essential component of the functional IL-12R on both T and natural killer (NK) cells. We previously observed that high affinity IL-12R were expressed on activated T and NK cells, but not B cells. Studies using flow cytometry and reverse transcription-polymerase chain reaction analysis showed that IL-12R beta chain was expressed on several human T, NK, and (surprisingly) B cell lines, but not on non-lymphohematopoietic cell lines. The Kit225/K6 (T cell) and SKW6.4 (B cell) lines were found to express the greatest amounts of IL-12R beta chain (800-2500 sites/cell); however, Kit225/K6 but not SKW6.4 cells bound IL-12. Similar to SKW6.4 B cells, activated tonsillar B lymphocytes expressed IL-12R beta chain but, consistent with previous results, did not display detectable IL-12 binding. Likewise, up to 72% of resting PBMC from normal volunteer donors expressed IL-12R beta, but did not bind measurable amounts of IL-12. These results indicate that expression of IL-12R beta is essential, but not sufficient, for expression of functional IL-12R. We speculate that expression of functional, high-affinity IL-12R may require the presence of a second subunit that is more restricted in its expression than IL-12R beta.

Characterization of anti-mouse IL-12 monoclonal antibodies and measurement of mouse IL-12 by ELISA.

Enzyme-linked immunosorbent assays (ELISAs) capable of quantitatively measuring pg/ml amounts of mouse IL-12 (moIL-12) were developed as an alternative to the current bioassay procedure used for the measurement of moIL-12. A panel of 40 rat anti-moIL-12 monoclonal antibodies were identified and tested for their ability to bind 125I-moIL-12. Two of the MAbs, 2B5 and 9A5, were able to capture 125I-moIL-12 in the presence of unlabelled moIL-12 p35 and moIL-12 p40, suggesting specificity for the moIL-12 p75 heterodimer. Western blot analysis confirmed that MAb 9A5 specifically recognized only moIL-12 p75. Using MAb 9A5, and an additional anti-moIL-12 p40 MAb 5D9, we developed quantitative ELISAs for the specific detection of moIL-12 p75 and p40, respectively. These ELISAs detect moIL-12 with a sensitivity of 40 pg/ml. Whereas the p40 ELISA detected three forms of moIL-12 (p40 monomer, p40 homodimer, and the heterodimer), the p75 ELISA only detected moIL-12 heterodimer. Neither of these assays crossreacted with a panel of additional cytokines. The levels of moIL-12 measured by the p75 ELISA and the bioassay were directly compared and found to correlate well. Therefore, the p75 ELISA represents an alternative to the bioassay for the measurement of moIL-12.

Administration of recombinant interleukin-12 to mice suppresses hematopoiesis in the bone marrow but enhances hematopoiesis in the spleen.

Although IL-12 has been reported to synergize with c-kit ligand (KL) in promoting hematopoietic stem cell proliferation in vitro, administration of recombinant mouse IL-12 (rIL-12) to normal mice caused a dose- and time-dependent anemia, leukopenia, and thrombocytopenia in vivo. Decreased numbers of bone marrow cells were recovered from the tibiae of IL-12-treated mice, and histologic examination of the marrow revealed a loss of mature neutrophils and red blood cell precursors. However, simultaneously with the suppression of hematopoiesis in the bone marrow, the IL-12-treated mice developed splenomegaly, which was largely caused by a marked enhancement of splenic extramedullary hematopoiesis of the erythroid, myeloid, and megakaryocytic lineages. These histologic observations were confirmed by colony-forming cell assays in which administration of IL-12 was shown to cause a time-dependent decrease in bone marrow CFU-GM, CFU-E, and BFU-E hematopoietic colony-forming cells while causing an increase in splenic CFU-GM and BFU-E colony-forming cells. All these effects were reversible upon cessation of IL-12 treatment. The observation that in IL-12-treated mice hematopoiesis was suppressed in the marrow but enhanced in the spleen suggests that myelosuppression was not caused by a direct effect of IL-12 on hematopoietic progenitors. It seems likely that myelosuppression was caused instead by an IL-12-induced alteration in the local environment of the marrow.

Structure-function analysis of the p35 subunit of mouse interleukin 12.

Mouse IL-12 acts on both mouse and human cells; human IL-12 acts only on human cells. This species specificity is determined by the p35 subunit of the IL-12 heterodimer. Since mouse and human p35 sequences are 60% identical, the determinants for the species specificity most likely residue in the nonhomologous sequences of mouse p35. To identify the regions on the p35 subunit interacting with the mouse IL-12 receptor, we constructed a series of chimeric mouse-human p35 molecules by replacing mouse sequences with the nonhomologous human counterparts. An IL-12 heterodimer containing a mouse-human p35 chimera with five residues changed in three discontinuous sites had drastically reduced (750-3000-fold) bioactivities on mouse cells. However, the competitive binding activity of the same mutant IL-12 heterodimer on mouse cells was only reduced 30-fold relative to wild-type IL-12. These findings therefore suggest that 1) the mouse p35 subunit participates in both receptor binding and signaling, 2) the mutations introduced into p35 affect signaling to a much greater extent than receptor binding, and 3) the five residues identified on p35 are required for interacting with the mouse, but not with the human IL-12 receptor and as such contribute extensively to the observed species specificity of IL-12.

Expression cloning of a human IL-12 receptor component. A new member of the cytokine receptor superfamily with strong homology to gp130.

A cDNA encoding a human IL-12R subunit was isolated by expression cloning. This subunit is a 662 amino acid type I transmembrane protein with an extracellular domain of 516 amino acids and a cytoplasmic domain of 91 amino acids. It is a member of the hemopoietin receptor superfamily and is most closely related over its entire length to gp130 and the receptors for granulocyte-CSF (G-CSF) and leukemia-inhibitory factor. When expressed in COS cells, this IL-12R subunit binds both human and murine IL-12 with an apparent affinity of 2 to 5 nM. The transfected COS cells express both monomers and disulfide-linked dimers or oligomers of the IL-12R subunit on their surface. However, unlike the IL-6-induced dimerization of gp130, the oligomerization of the IL-12R subunit is not dependent on binding of IL-12. Only the IL-12R subunit dimers/oligomers but not the monomers bind IL-12 with an affinity of 2 to 5 nM. A polyclonal antiserum raised against this receptor subunit specifically inhibits IL-12-induced proliferation of PHA-activated PBMC. The data are consistent with the hypotheses that 1) a dimer/oligomer of the cloned IL-12R subunit (IL-12R-beta) represents the low affinity IL-12 binding site identified on human lymphoblasts, 2) the cloned receptor subunit is involved in IL-12 signal transduction, and 3) an additional, as of yet unidentified subunit is required to generate a high affinity IL-12R complex.

Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo.

IL-12 is a heterodimeric cytokine that has been shown to enhance natural killer (NK) and cytotoxic T lymphocyte (CTL) responses, and to induce IFN-gamma production in vitro. In this study, we have examined the effects in vivo of administering purified murine rIL-12 to normal mice. Daily injections of rIL-12 i.p. (1 ng to 10 micrograms/day) caused dose-dependent enhancement of NK cell lytic activity in the spleens and livers of treated mice. Histologic examination of the livers of IL-12-treated mice revealed focal mononuclear cell infiltrates, and flow cytometry studies indicated that the livers of IL-12-treated mice contained increased numbers of NK cells, CD8+ T cells, and monocytes. Liver and splenic lymphoid cells from IL-12-treated mice, unlike liver and splenic lymphoid cells from control mice, spontaneously secreted IFN-gamma in vitro, suggesting that they had been induced by IL-12 to produce IFN-gamma in vivo. Consistent with this, IFN-gamma could be detected in the serum of IL-12-treated mice. In mice which had been immunized by footpad injection of allogeneic splenocytes, daily administration of rIL-12 i.p. was shown to enhance the specific CTL response in the draining lymph nodes. Thus, these studies demonstrate that IL-12 can enhance NK and CTL activity and induce IFN-gamma production in vivo, as well as in vitro, and suggest possible mechanisms by which IL-12 may exert therapeutic effects in the treatment of some tumors and infectious diseases.

Antitumor and antimetastatic activity of interleukin 12 against murine tumors.

It has recently been demonstrated that in vivo administration of murine interleukin 12 (IL-12) to mice results in augmentation of cytotoxic natural killer (NK)/lymphocyte-activated killer cell activity, enhancement of cytolytic T cell generation, and induction of interferon gamma secretion. In this study, the in vivo activity of murine IL-12 against a number of murine tumors has been evaluated. Experimental pulmonary metastases or subcutaneous growth of the B16F10 melanoma were markedly reduced in mice treated intraperitoneally with IL-12, resulting in an increase in survival time. The therapeutic effectiveness of IL-12 was dose dependent and treatment of subcutaneous tumors could be initiated up to 14 d after injection of tumor cells. Likewise, established experimental hepatic metastases and established subcutaneous M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity. Local peritumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors. IL-12 was as effective in NK cell-deficient beige mice or in mice depleted of NK cell activity by treatment with antiasialo GM1, suggesting that NK cells are not the primary cell type mediating the antitumor effects of this cytokine. However, the efficacy of IL-12 was greatly reduced in nude mice suggesting the involvement of T cells. Furthermore, depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12. These results demonstrate that IL-12 has potent in vivo antitumor and antimetastatic effects against murine tumors and demonstrate as well the critical role of CD8+ T cells in mediating the antitumor effects against subcutaneous tumors.