Regulation of human cytolytic lymphocyte responses by interleukin-12.

IL-12 is a heterodimeric cytokine which has been shown to cause the proliferation of activated T and NK cells, to enhance the lytic activity of NK cells, and to induce IFN-gamma production by resting and activated T and NK cells. We previously reported that IL-12 could synergize with IL-2 to activate human LAK cells in the presence of hydrocortisone but that IL-12 alone was inactive. We herein show that in the absence of hydrocortisone, IL-12 by itself can activate human LAK cells. IL-12-induced LAK cell activity was mediated predominantly by CD56+ lymphocytes. Activation of LAK cells by IL-12 appeared to be independent of IL-2 since it was not inhibited by neutralizing anti-human IL-2. However, IL-12- and IL-2-induced LAK cell activation could be partially inhibited by anti-human TNF-alpha. Moreover, IL-12 produced in situ appeared to play a role in IL-2-induced LAK cell activation since rat monoclonal antibodies to human IL-12 could partially inhibit the generation of LAK cells in response to IL-2. In addition to its effects on LAK cell responses, IL-12 could facilitate specific allogeneic human CTL responses. However, IL-12-facilitated CTL responses were blocked by neutralizing anti-human IL-2 indicating a requirement for IL-2 produced in situ. The ability of IL-12 to facilitate both nonspecific LAK and specific CTL responses suggests that it may be useful as a therapeutic agent against some tumors and infectious diseases.     

Characterization of monoclonal antibodies against human leukocyte 5-lipoxygenase

Four mouse monoclonal IgG1 antibody-producing cell lines (5LO-1, 5LO-2, 5LO-3, 5LO-4), produced against highly purified human leukocyte 5-lipoxygenase have been characterized. The monoclonal antibodies produced by these cell lines exhibited differential reactivity against 5-lipoxygenase as determined by ELISA and immunoprecipitation analyses. Monoclonal antibodies 5LO-2 and 5LO-3 inhibited the activity of recombinant human leukocyte 5-lipoxygenase in a dose-dependent manner. This inhibition was selective for 5-lipoxygenase activity since these monoclonal antibodies did not inhibit human leukocyte 15-lipoxygenase or porcine leukocyte 12-lipoxygenase.     

Lymphocyte mitogenesis induced by monoclonal antibodies to the T3 complex. Differential modulation by human IgG

Murine monoclonal antibodies OKT3 (IgG2), 64.1 (IgG2), and Leu 4 (IgG1) react with a common membrane antigen on human T cells and induce potent mitogenesis at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml, respectively. Human serum inhibits the mitogenic effect of antibodies OKT3 and 64.1, but not that of Leu 4. The inhibitor in serum has been identified as immunoglobulin G (IgG) as evidenced by the ability of anti-human IgG-Sepharose affinity columns to retain the inhibitory activity. Various immunoglobulin classes and subclasses obtained from human myelomas differ in their ability to inhibit the OKT3-induced activation. The best inhibition is obtained with the IgG subclasses IgG1 and IgG3, followed by IgG2; IgG4, IgM, and IgA have little if any effect. None of the IgG subclasses inhibit the Leu 4-induced mitogenesis. Indomethacin as well as supernatants containing interleukin 2 (IL-2) can reverse the inhibitory effects of IgG. Prostaglandins (PGE1 and PGE2) inhibit both the OKT3- and Leu 4-induced mitogenesis, thus lacking the selectivity seen with IgG. Since stimulation by the monoclonal antibodies requires the participation of monocytes, an interpretation consistent with the present data is that IgG stimulates monocytes via its Fc portion to release prostaglandins and/or other suppressor factors via an indomethacin-sensitive pathway. The inability of IgG to inhibit Leu 4-induced mitogenesis may therefore relate to an inability of the monocyte subpopulation, which mediates the Leu 4 response, to secrete suppressor factors. These data suggest a potential value of the mitogenic monoclonal antibodies as probes in studying monocyte heterogeneity and T-cell-monocyte interactions.     

Monoclonal autoantibodies with interleukin 3-like activity derived from a MRL/lpr mouse

Hybridomas that secrete monoclonal antibodies with interleukin 3 (IL-3)-like activity were established from spleen cells of a nonimmunized autoimmune MRL/lpr mouse. Five of the monoclonal antibodies thus obtained bound selectively to IL-3-dependent cells and supported their growth. These monoclonal antibodies inhibited the binding of IL-3 to FDC-P2 cells and vice versa. Thus, these antibodies were probably directed to IL-3 receptor sites, or at least to some cell surface proteins related to the growth of the IL-3-dependent cells. These MRL/lpr-derived monoclonal antibodies reacted strongly with cells from bone marrow, spleen, and lymph node of MRL/lpr mice, but minimally with such cells of MRL/+ or BALB/c mice. The findings were consistent with our earlier suggestion that the IL-3-like activity in MRL/lpr sera is not caused by IL-3 itself but is associated with IgG that is probably an autoantibody directed to the IL-3 receptor.     

Spred-1 negatively regulates interleukin-3-mediated ERK/mitogen-activated protein (MAP) kinase activation in hematopoietic cells

Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form of Spred-1, DeltaC-Spred, in IL-3- and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, DeltaC-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas DeltaC-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCF-induced but also IL-3-induced ERK activation.     

Novel antagonist antibody to TLR3 blocks poly(I:C)-induced inflammation in vivo and in vitro

Toll-like receptor 3 (TLR3) binds and signals in response to dsRNA and poly(I:C), a synthetic double stranded RNA analog. Activation of TLR3 triggers innate responses that may play a protective or detrimental role in viral infections or in immune-mediated inflammatory diseases through amplification of inflammation. Two monoclonal antibodies, CNTO4685 (rat anti-mouse TLR3) and CNTO5429 (CDRs from CNTO4685 grafted onto a mouse IgG1 scaffold) were generated and characterized. These mAbs bind the extracellular domain of mouse TLR3, inhibit poly(I:C)-induced activation of HEK293T cells transfected with mTLR3, and reduce poly(I:C)-induced production of CCL2 and CXCL10 by primary mouse embryonic fibroblasts. CNTO5429 decreased serum IL-6 and TNFα levels post-intraperitoneal poly(I:C) administration, demonstrating in vivo activity. In summary, specific anti-mTLR3 mAbs have been generated to assess TLR3 antagonism in mouse models of inflammation.     

Characterization of mouse macrophage differentiation antigens by monoclonal antibodies

Monoclonal rat antibodies to mouse macrophage antigens were prepared. For immunization phagocytic cells in the spleens of mice recovering from sublethal irradiation were used. Specificities of the monoclonal antibodies obtained were determined on cells of normal mouse cell populations as well as on cells of a panel of mouse cell lines. In an attempt to monitor expression of differentiation-related antigens two models of in vitro-induced macrophage differentiation were used: differentiation of cells of the myeloblast line Ml; CSF-1-induced differentiation of bone marrow cells. The results obtained clearly show that during maturation from undifferentiated to highly differentiated cells of the macrophage lineage expression of antigens recognized by the MIV 38, MIV 55, MV 87, and MV 114 monoclonal antibodies is enhanced. At the same time, expression of antigens recognized by the MIV 52, MIV 113, and MIV 116 monoclonal antibodies diminishes at a similar rate. The suitability of these monoclonal antibodies for the characterization of differentiation states of mouse macrophages is discussed.     

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Interleukin 2 induces interferon alpha/beta production in mouse bone marrow cells

We have previously reported that mouse bone marrow (BM) cells stimulated with alloantigen produce cytotoxic effector T-cell activity and produce interferon (IFN-)alpha/beta. In this report we show evidence suggesting that interleukin 2 (IL-2) may play a role in this IFN-alpha/beta production by alloantigen-stimulated BM cells. Alloantigen-induced IFN production by bone marrow cells was completely inhibited when cultures were supplemented with antisera to IL-2. Cell-free supernatants obtained at 2 days from cultures containing C57BL/6 BM cells and irradiated DBA/2J spleen cells were also shown to contain low levels of IL-2 activity and induced significant IFN production in fresh BM cells. Different IL-2 preparations were tested for their ability to induce IFN-alpha/beta production in mouse BM cells. Mouse BM cells cultured with recombinant human IL-2 or highly purified mouse IL-2 produced high levels of IFN-alpha/beta activity after 2-3 days of culture with significant IFN activity being detected as early as 24 hr of culture. IL-2-induced IFN-alpha/beta production was partially resistant to irradiation. In contrast, irradiated (2000 rad) bone marrow cells failed to produce any IFN when cultured with alloantigen in the absence of IL-2. T-cell-depleted BM cells or BM cells obtained from C57BL/10 nude mice produced high levels of IFN-alpha/beta following stimulation with IL-2. In addition, bone marrow cells depleted of Ia+, Qa 5+, or Asialo GM+1 cells produced IFN in response to IL-2. Thus, neither T cells nor NK cells are required for IL-2-induced IFN-alpha/beta production by BM cells. The action of IL-2 on bone marrow cells to induce IFN production was mediated by the classical IL-2 receptor, since monoclonal antibodies to the IL-2 receptor present on T cells blocked this response and since bone marrow cells depleted of IL-2 receptor-bearing cells failed to produce IFN when cultured with IL-2. These results suggest that non-T cells resident in the BM have receptors for IL-2 and can produce IFN-alpha/beta upon stimulation by IL-2. Since IFN has been shown to affect different aspects of hematopoiesis, the production of IFN by BM cells stimulated by IL-2 may be important in the control of hematopoiesis. In addition, IL-2-induced IFN production may play a role in graft-versus-host disease.     

Different effects of IL-2 addition or antibody crosslinking on T-cell subset stimulation by CD3 antibodies

The effect of exogenous recombinant interleukin-2 (IL-2) or of antibody crosslinking on the activation of human T-cell subsets by IgG2a (OKT3/BMA030), IgG1 (Leu4 and UCHT1), or IgG2b (BMA031) anti-T3 antibodies (CD3) was investigated. In so-called nonresponder cultures as well as in monocyte-depleted cell cultures addition of IL-2 increased the CD3-induced activation and proliferation of T4 and T8 cell subsets. Relatively more T8 than T4 cells were stimulated by antibody binding and IL-2. Crosslinking the cell-bound CD3 antibodies by plastic bound goat anti-mouse antibodies activated both T-cell subsets optimally and increased the IL-2 production of the IgG1-CD3 stimulated cultures. The data show that T cells (T8 greater than T4) can be stimulated by CD3 antibody binding and IL-2, but that crosslinking the cell-bound CD3 antibodies is crucial for optimal T4 cell stimulation and IL-2 production.     

Reciprocal regulation of 17β-estradiol, interleukin-6 and interleukin-8 during growth and progression of epithelial ovarian cancer

Estrogens have been associated with risk for epithelial ovarian cancer (OVCA). Both IL-6 and IL-8 are also likely involved in the progression of OVCA. In order to discover the underline molecular mechanism, we investigated the modulation of estrogen and two cytokines in the growth and progression of epithelial OVCA. In these studies, the effect of 17β-estradiol (E₂) on the expression levels of IL-6, IL-8 and their receptors was investigated. The effect of IL-6 and IL-8 on activation of estrogen-responsive promoter as well as estrogen receptor (ER)α and ERβ expression was also analyzed. Gene expression profile analysis revealed that CAOV-3 and OVCAR-3 cells, which express ER, IL-6 and IL-8 receptors, are suitable model for this study. We found that E₂ not only enhanced IL-6 and IL-8 production via NF-κB signaling pathway, but also modulated their respective receptor expression. Tamoxifen (Txf), an ER antagonist, completely abolished E₂-stimulated cell growth and the expression of IL-6 and IL-8. IL-6/IL-8-induced cell proliferation was completely blocked by their specific neutralizing antibodies, which partially inhibited E₂-induced cell growth. In the absence of estrogen, both cytokines activated estrogen-responsive promoter, which was completely blocked by Txf, and caused a dose-dependent ERα increase and ERβ decrease. Pretreatment of OVCAR-3 with p38 MAPK, MEK1/2 or ErbB2 MAPK inhibitors, respectively, blocked IL-6-mediated induction of estrogen-responsive promoter while Src inhibitor blocked IL-8-induced activation of estrogen-responsive promoter. These results provide a novel mechanism that estrogens, IL-6 and IL-8 may form a common amplifying signaling cascade to modulate OVCA growth and progression. Estrogen-induced OVCA proliferation is partially occurring via enhanced IL-6 and IL-8 production and modulated their receptors, and IL-6/IL-8 could also promote OVCA growth through an ERα pathway.     

Human recombinant IL-4 induces activated B lymphocytes to produce IgG and IgM

In this report, we describe a novel biologic activity of IL-4 namely, its ability to induce activated human B cells to produce IgM. Staphylococcus aureus Cowan I-activated blasts prepared from high density tonsil B cells were found to secrete IgG and IgM, but no IgE, when cultured in the presence of rIL-4. The differentiating activity of rIL-4 was totally blocked by a neutralizing anti-IL-4 antiserum, therefore demonstrating that the IgG/IgM-inducing activity of rIL-4 was an intrinsic property of IL-4. rIL-4 was only minimally inducing Ig production of blasts prepared from low density B cells, whereas it induced B cell blasts prepared from high density B cells to secrete a high amount of Ig. Delayed additions of the neutralizing anti-IL-4 antiserum demonstrated that a 48-h contact between IL-4 and B cell blasts was required for optimal Ig production. The IL-4-mediated IgG and IgM production was neither suppressed by IFN-gamma nor by anti-CD23 mAb 25, whereas these agents have been shown earlier to inhibit IgE production of enriched B cells cultured in the presence of IL-4. These data indicate that the IgG/IgM-inducing activity of IL-4 is not regulated like the IL-4-induced IgE production by enriched B cells.     

Identification of human, mouse, and rat retinoic acid receptor alpha using monoclonal antibodies.

Monoclonal antibodies that recognize the human, mouse, and rat retinoic acid receptor alpha (RAR alpha) protein have been generated using synthetic peptides. Less well-characterized monoclonal antibodies were also generated against the RAR beta and RAR gamma proteins. Monoclonal antibodies of the IgG1 (R alpha 10) and IgG2a (R alpha 13) isotypes effectively and specifically recognize both the human and mouse RAR alpha protein. Preincubation of the antibodies with the synthetic RAR alpha peptide, but not with the RAR beta or RAR gamma peptides, blocked recognition of the approximately 55 kDa RAR alpha protein on western blots. These monoclonal antibodies also detected differing levels of RAR alpha in various rat tissues. These monoclonal antibodies will serve as powerful reagents to study the structure and regulation of the retinoic acid receptor protein.     

Natural cytotoxic cell-specific cytotoxic factor produced by IL-3-dependent basophilic/mast cells. Relationship to TNF

The murine IL-3-dependent mast cell line, PT18-A17, and the rat basophilic leukemia cell line, RBL-2H3, were found to mediate natural cytotoxic (NC) activity via the release of a soluble factor which specifically lysed NC-sensitive WEHI-164 but not NK-sensitive YAC-1 tumor cells. The release of this NC cell-specific cytotoxic factor was enhanced by triggering of both types of cells via IgE receptor bridging. This factor had activity on TNF-sensitive but not TNF-resistant cell lines and could be neutralized by two independently produced polyclonal anti-mouse TNF antisera. It was not neutralized by antibodies against mouse IFN-alpha/beta or IFN-gamma. Moreover, it was not neutralized by a monoclonal or a polyclonal anti-human TNF, demonstrating that the rodent TNF differed antigenically from human TNF. These results indicate that the cytotoxic factor released from a murine IL-3-dependent mast cell line and from a rat basophilic leukemia cell line is immunologically and functionally related to murine TNF.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Human monoclonal antibodies to Pseudomonas aeruginosa produced by EBV-transformed cells

Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD50) values were 0.5, 2.6, and 3.1 micrograms immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

Identification of a novel protein capable of interacting with the IL-3 receptor

IL-3 has numerous functions in hematopoiesis yet its receptor has not been fully characterized. We have developed two mAb, 4G8 and 2F2, that markedly inhibited IL-3-dependent proliferation whereas only marginally affecting IL-2 or IL-4-induced proliferation. On Western blots, both antibodies identified the same protein, which varied in size from 115 to 145 kDa in six cell lines tested. The 4G8/2F2 Ag was detected at moderate density, on a wide variety of cells including IL-3-dependent cell lines and T lymphocytes. Radioligand binding studies revealed that 4G8, but not 2F2, could inhibit the binding of 125I-IL-3 to the high affinity IL-3R. These data suggest that the mAb 4G8 and 2F2 recognize different epitopes on the same Ag, and suggest furthermore that the inhibition of IL-3-dependent proliferation mediated by 2F2, in particular, does not occur via inhibition of ligand binding. Neither antibody showed an enhanced level of fluorescent staining of Cos 7 cells transfected with the low affinity IL-3R cDNA. In addition, 4G8 did not inhibit IL-3 binding to L cells transfected with the cloned IL-3R or IL-4R despite the fact that 4G8 was expressed on these cells. These data suggest that the 4G8/2F2 Ag is a unique cell surface protein that can interact with the endogenous functional IL-3R.