Protection from RNA and DNA viruses by IL-32

Several studies have documented a proinflammatory role for IL-32, which induces IL-1α, IL-1β, IL-6, TNF, and chemokines via NF-κB, p38MAPK, and AP-1. However, IL-32 also participates in the responses to infection with viruses such as HIV-1 and influenza. In this study, we explored these antiviral properties of IL-32. Vital staining assays demonstrated that low concentrations (5-10 ng/ml) of rIL-32γ protected epithelial WISH cells from vesicular stomatitis virus-induced cell death. By lactate dehydrogenase assays, treatment with IL-32γ resulted in a 3- to 4-fold decrease in viral load. Specific silencing of IL-32 revealed that the antiviral responses triggered by the synthetic analogs of ssRNA viruses (polyuridine) and dsRNA viruses (polyinosinic-polycytidylic acid) were significantly weaker (2- to 3-fold more virus) in WISH cells in the absence of IL-32. Importantly, we discovered that the polyinosinic-polycytidylic acid-induced increase in production of IFN-α in human PBMC was nearly completely abolished when IL-32 was silenced. Moreover, we observed that IL-32 antagonizes the DNA virus HSV-2 in epithelial Vero cells as well as in human umbilical cord endothelial cells, as production of HSV-2 increased 8-fold upon silencing of IL-32 (p < 0.001). Mechanistically, we found that IL-32 used the PKR-eIF-2α as well as the MxA antiviral pathways. Unexpectedly, a considerable part of the antiviral properties of IL-32 was not dependent on IFNs; specific blockade of IFN activity reduced the antiviral properties of IL-32 only moderately. In conclusion, these data suggest a central role for IL-32 in the immune response to RNA and DNA viruses, which may be exploitable for clinical use in the future.     

IL-32γ induces chemotaxis of activated T cells via dendritic cell-derived CCL5

Interleukin (IL)-32 has been associated with a variety of inflammatory diseases including rheumatoid arthritis, vasculitis and Crohn’s disease. We have previously reported that IL-32γ, the IL-32 isoform with the highest biological activity, could act as an immune modulator through regulation of dendritic cell (DC) functions in immune responses. Cell locomotion is crucial for induction of an effective immune response. In this study, we investigated the effect and underlying mechanisms of IL-32γ on recruitment of T cells. IL-32γ upregulated the expression of several chemokines including CCL2, CCL4, and CCL5 in the DCs. In particular, IL-32γ significantly increased CCL5 expression in a dose-dependent manner. Treatment with JNK and NF-κB inhibitors suppressed IL-32γ-induced CCL5 expression in DCs, indicating that IL-32γ induced CCL5 production through the JNK and NF-κB pathways. Furthermore, supernatants from IL-32γ-treated DCs showed chemotactic activities controlling migration of activated CD4⁺ and CD8⁺ T cells, and these activities were suppressed by addition of neutralizing anti-CCL5 antibody. These results show that IL-32γ effectively promotes migration of activated T cells via CCL5 production in DCs. The chemotactic potential of IL-32γ may explain the pro-inflammatory effects of IL-32 and the pathologic role of IL-32 in immune disorders such as rheumatoid arthritis.     

T cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue

Genetic studies with immunocompetent mice show the importance of both T cells and gamma interferon (IFN-γ) for survival of a measles virus (MV) challenge; however, the direct role of T cells and IFN-γ within the MV-infected brain has not been addressed. Organotypic brain explants represent a successful ex vivo system to define central nervous system (CNS)-specific mechanisms of leukocyte migration, activation, and MV clearance. Within the heterogeneous, brain-derived, primed leukocyte population which reduced MV RNA levels in brain explants by 60%, CD3 T cells are the active antiviral cells, as purified CD3-positive cells are highly antiviral and CD3-negative leukocytes are unable to reduce the viral load. Neutralization of CCL5 and CXCL10 decreases leukocyte migration to areas of infection by 70%. However, despite chemokines directing the migration of T cells to infected neurons, chemokine neutralization revealed that migration is not required for viral clearance, suggesting a cytokine-mediated antiviral mechanism. In accordance with our hypothesis, the ability of leukocytes to clear the virus is abrogated when explants are treated with anti-IFN-γ neutralizing antibodies. IFN-γ applied to infected slices in the absence of primed leukocytes reduces the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear MV. Secretion of IFN-γ is stimulated by interleukin-12 (IL-12) in the brain, as neutralization of IL-12 results in loss of antiviral activity and stimulation of leukocytes with IL-12/IL-18 enhances their immune effector function of viral clearance. MV-primed leukocytes can reduce both West Nile and mouse hepatitis viral RNAs, indicating that cytokine-mediated viral clearance occurs in an antigen-independent manner. The IFN-γ signal is transduced within the brain explant by the Jak/STAT signaling pathway, as inhibition of Jak kinases results in a loss of antiviral activity driven by either brain-derived leukocytes or recombinant IFN-γ. These results reveal that primed T cells directly act to clear MV infection of the brain by using a noncytolytic IL-12- and IFN-γ-dependent mechanism in the CNS and that this mechanism relies upon Jak/STAT signaling.     

IL-32gamma induces the maturation of dendritic cells with Th1- and Th17-polarizing ability through enhanced IL-12 and IL-6 production

IL-32, a newly described multifunctional cytokine, has been associated with a variety of inflammatory diseases, including rheumatoid arthritis, vasculitis, and Crohn's disease. In this study, we investigated the immunomodulatory effects of IL-32γ on bone marrow-derived dendritic cell (DC)-driven Th responses and analyzed the underlying signaling events. IL-32γ-treated DCs exhibited upregulated expression of cell-surface molecules and proinflammatory cytokines associated with DC maturation and activation. In particular, IL-32γ treatment significantly increased production of IL-12 and IL-6 in DCs, which are known as Th1- and Th17-polarizing cytokines, respectively. This increased production was inhibited by the addition of specific inhibitors of the activities of phospholipase C (PLC), JNK, and NF-κB. IL-32γ treatment increased the phosphorylation of JNK and the degradation of both IκBα and IκBβ in DCs, as well as NF-κB binding activity to the κB site. The PLC inhibitor suppressed NF-κB DNA binding activity and JNK phosphorylation increased by IL-32γ treatment, thereby indicating that IL-32γ induced IL-12 and IL-6 production in DCs via a PLC/JNK/NF-κB signaling pathway. Importantly, IL-32γ-stimulated DCs significantly induced both Th1 and Th17 responses when cocultured with CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb abolished the secretion of IFN-γ in a dose-dependent manner; additionally, the blockage of IL-1β and IL-6, but not of IL-21 or IL-23p19, profoundly inhibited IL-32γ-induced IL-17 production. These results demonstrated that IL-32γ could effectively induce the maturation and activation of immature DCs, leading to enhanced Th1 and Th17 responses as the result of increased IL-12 and IL-6 production in DCs.     

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-β (IFN-β) or interferon-γ (IFN-γ). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-γ were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-β. When IFN-β-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-γ-treated cells. LPS and MTP also upregulated IFN-γ-mediated IDO activity when suboptimal amounts of IFN-γ were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1α (IL-1α), along with either maximum-stimulating amounts of IFN-β or suboptimal amounts of IFN-γ, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1α or IL-1β was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1α was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1α abolished the upregulatory effect of exogenous IL-1α, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1α, upregulation of IDO activity by these agents is independent of IL-1α production and may be mediated through distinct pathways.     

Synergy between TLR3 and IL-18 promotes IFN-γ dependent TRAIL expression in human liver NK cells

Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. However, intra-hepatic NK cells’ ability to respond to virus is still mostly unknown. Our results show that the synthetic dsRNA polyinosinic–polycytidylic acid (poly I:C), a mimic of a common product of viral infections, activates NK cells directly in the context of cytokines found in the liver, i.e.: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-γ production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-γ blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-γ is an autocrine differentiation factor for these cells. A better understanding of the intra-hepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.     

STAT1-independent control of a neurotropic measles virus challenge in primary neurons and infected mice

Neurons are chiefly nonrenewable; thus, cytolytic immune strategies to clear or control neurotropic viral infections could have lasting neurologic consequences. IFN-γ is a potent antiviral cytokine that is critical for noncytolytic clearance of multiple neurotropic viral infections, including measles virus (MV); however, the downstream pathways through which IFN-γ functions in neurons have not been defined. Unlike most cell types studied to date in which IFN-γ affects gene expression via rapid and robust activation of STAT1, basal STAT1 levels in primary hippocampal neurons are constitutively low, resulting in attenuated STAT1 activation and consequently slower kinetics of IFN-γ-driven STAT1-dependent gene expression. Given this altered expression and activation of STAT1 in neurons, we sought to determine whether STAT1 was required for IFN-γ-mediated protection from infection in neurons. To do so, we evaluated the consequences of MV challenge of STAT1-deficient mice and primary hippocampal neurons explanted from these mice. Surprisingly, the absence of STAT1 did not restrict the ability of IFN-γ to control viral infection either in vivo or ex vivo. Moreover, the canonical IFN-γ-triggered STAT1 gene expression profile was not induced in STAT1-deficient neurons, suggesting that IFN-γ regulates neuronal STAT1-independent pathways to control viral replication.     

IL-6 augments Fc IgE receptor (Fc epsilon RII/CD23) expression on human monoblastic/monocytic cell lines U937, THP-1, and Mono-Mac-6 but not on blood monocytes. Regulatory effects of IL-4 and IFN-gamma.

Human monoblastic/monocytic leukemia cell lines U937, THP-1, Mono-Mac-6, and blood monocytes were incubated with various concentrations of human rIL-6 and other cytokines and analyzed for their capacity to bind several anti-Fc epsilon RII/CD23 mAb. A marked and dose-dependent increase in the percentage of CD23+ cells, as well as in the mean channel fluorescence intensity, as demonstrated by FACS analysis, was noted after 8- to 72-h incubation of U937 cells with 1 to 1000 U/ml of human rIL-6. Furthermore, rIL-4 synergized with rIL-6 and rIFN-tau in augmenting the Fc epsilon RII expression on U937 cells, whereas rIFN-tau and rIL-6 showed rather additive effects. The enhancement of CD23 expression on IL-6-treated U937 cells was blocked by anti-IL-6 antibodies. Northern blot analysis, employing cDNA probes for Fc epsilon RII, showed that U937 cells contain Fc epsilon RII-specific mRNA. The level of Fc epsilon RII-encoding mRNA was evidently increased by treatment of U937 cells with human rIL-6, rIL-4, or with rIL-6 + rIL-4. The expression of CD23 on THP-1 and Mono-Mac-6 cells was increased slightly by rIL-6 and markedly by rIL-4, rIFN-tau, or a mixture of them. Approximately 14% of blood monocytes, isolated from apparently healthy donors, constitutively possess Fc epsilon RII. In contrast to the cell lines, the Fc epsilon RII density and the percentage of blood monocytes bearing Fc epsilon RII was not augmented by IL-6. Furthermore, rIL-6, and more evidently rIFN-tau, down-regulate rIL-4-driven Fc epsilon RII expression on monocytes but not on monocytic cell lines. Our findings point to differences in the capability of mononuclear phagocytes to respond to cytokine treatment, which may be differentiation dependent, and suggest separate regulatory pathways.     

Gamma interferon controls mouse polyomavirus infection in vivo

Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.     

Interleukin-1 inhibits hepatitis C virus subgenomic RNA replication by activation of extracellular regulated kinase pathway

Interleukin-1 (IL-1) plays an important role in the inflammatory process. Some studies have demonstrated that IL-1 production was impaired in patients with chronic infections of hepatitis C virus (HCV), implying that IL-1 may play a role in viral clearance. Using an HCV subgenomic replicon cell line, we demonstrate that IL-1 can effectively inhibit HCV subgenomic RNA replication and viral protein expression, suggesting that IL-1 has direct antiviral activity. The inhibitory effect is associated with the extracellular regulatory kinase (ERK) activation. In addition, we also show that IL-1 can induce one of the interferon-stimulated genes (ISGs), 1-8U, which exhibits antiviral activity. However, it has no effect on the other ISG, 6-16, suggesting that IL-1 induces novel antiviral pathways within a cell.     

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.     

白细胞介素21对SHIV感染的恒河猴外周血CD8~＋ T细胞分泌γ干扰素的影响

目的研究白细胞介素21（interleukin 21,IL-21）对SHIV感染CD8＋T细胞分泌干扰素γ（interferon-γ,IFN-γ）的影响。方法从SHIV/恒河猴模型外周血中分选出CD8＋T细胞,加入IL-21诱导培养,应用ELISA方法检测细胞培养上清液中IFN-γ浓度,RT-PCR方法检测细胞中IFN-γmRNA的表达水平,流式细胞术检测分泌IFN-γ的CD8＋T细胞所占的百分比。结果 10 ng/mL IL-21明显促进CD8＋T细胞分泌IFN-γ（P〈0.05）,IFN-γmRNA的表达明显升高,4 h为刺激CD8＋T细胞胞内IFN-γ合成的最佳时间。结论 IL-21对CD8＋T细胞分泌IFN-γ有促进作用。     

Activity of CD4+ T-cell clones of type 1 and type 2 in generation of influenza virus-specific cytotoxic responses in vitro

The activity of distinct CD4+ T-helper cell (Th) clones in promoting secondary A/PR/8/34/Mt.S.(H1N1) (A/PR8) influenza virus-specific, class I-restricted cytotoxic T-lymphocyte (CTL) responses in vitro was examined. CD8+ T cells which had been purified by fluorescence-activated cell sorter from spleen cells of A/PR8-primed mice were used as responders. On their own, purified CD8+ T cells were unable to generate cytotoxic activity upon in vitro culture with A/PR8-infected stimulator cells. Significant cytotoxic activity was generated in cultures that were additionally supplemented with A/PR8-specific Th clones or cell-free supernatant from these clones. Although there were large differences among individual Th clones in this function, Th clones of type 1 (Th1) promoted, on average, significantly stronger cytotoxic responses than Th clones of type 2 (Th2). The differences in promotion of a cytotoxic response correlated with the amount of interleukin-2 (IL-2) or IL-4 secreted by individual Th clones. These two lymphokines accounted for the CTL-promoting activity of the respective Th clones, since addition of recombinant IL-2 (IL-2) or rIL-4 to Th-free cultures substituted fully for the respective Th clones. As observed with Th clones, rIL-2 was significantly more effective than rIL-4 in promoting a cytotoxic response. When used in combination, Th2 clones had an antagonistic effect on the generation of a CTL response by Th1 clones. This effect could be partially transferred with cell-free supernatant from activated Th2 clones and could be reversed by addition of excess rIL-2. Both consumption of IL-2 by Th2 and secretion of an inhibitory factor(s) appear to be involved in this phenomenon.     

Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection

NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-γ in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-γ production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56(bright) NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-γ. Blockade of IL-10 +/- TGF-β restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-γ persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/- TGF-β blockade.     

Interleukin-2-induced production of interferon-gamma by resting human T cells and large granular lymphocytes: requirement for accessory cell factors, including interleukin-1

Between 5 and 20% of normal human lymphocytes were found to synthesize interferon-gamma (IFN-gamma) in primary cultures with recombinant interleukin-2 (rIL-2). After 22 hr, IFN-gamma-producing cells included CD5+ T lymphocytes, CD16+ large granular lymphocytes (LGL), and a population of CD5-, CD16- blast cells. Only a small proportion (0-7%) of IFN-gamma-synthesizing cells expressed HLA-DR. The production of IFN-gamma by all rIL-2-responding lymphocyte subsets was shown to require the presence of DR+ accessory cells, probably including nonadherent, esterase-negative monocytes and/or dendritic cells. Accessory cell function in lymphocyte preparations depleted of DR+ cells, or in purified (greater than or equal to 95%) suspensions of LGL, was fully replaced either by addition of 2% autologous, adherent monocytes or by monocyte culture supernatant. The activity of monocyte supernatant was greatly reduced by treatment with antiserum specific for human interleukin-1 beta (IL-1 beta), although a combination of rIL-1 beta and rIL-2 failed to stimulate IFN-gamma production in DR- lymphocytes. These results indicate that rIL-2-induced IFN-gamma synthesis in both T cells and LGL requires the synergistic activity of IL-1, and possibly of one or more other monokines, as yet unidentified.     

Lipocalin-2-induced cytokine production enhances endometrial carcinoma cell survival and migration

Lipocalin-2 (Lcn-2) is an acute-phase protein that has been implicated in diverse physiological processes in mice, including: apoptosis, ion transport, inflammation, cell survival, and tumorigenesis. This study characterized the biological activity of Lcn-2 in human endometrial carcinoma cells (RL95-2). Exposure of RL95-2 cells to Lcn-2 for >24 h reduced Lcn-2-induced cell apoptosis, changed the cell proliferation and up-regulated cytokine secretions, including: interleukin-8 (IL-8), inteleukin-6 (IL-6), monocyte chemotatic protein-1 (MCP-1) and growth-related oncogene (GRO). However, IL-8 mRNA and protein levels were dramatically increased in Lcn-2-treated RL95-2 cells. To determine the IL-8 effect on Lcn-2-treated RL95-2 cells was our major focus. Adding recombinant IL-8 (rIL-8) resulted in decreased caspase-3 activity in Lcn-2-treated cells, whereas the addition of IL-8 antibodies resulted in significantly increased caspase-3 activity and decreased cell migration. Data indicate that IL-8 plays a crucial role in the induction of cell migration. Interestingly, Lcn-2-induced cytokines, secretion from RL95-2 cells, could not show the potent cell migration ability with the exception of IL-8. We conclude that Lcn-2 triggered cytokine secretions to prevent RL95-2 cells from undergoing apoptosis and subsequently increased cell migration. We hypothesize that Lcn-2 increased cytokine secretion by RL95-2 cells, which in turn activated a cellular defense system. This study suggests that Lcn-2 may play a role in the human female reproductive system or in endometrial cancer.     

Both B151-T cell replacing factor 1 and IL-5 regulate Ig secretion and IL-2 receptor expression on a cloned B lymphoma line

In the present study, we have demonstrated that both B151-T cell-replacing factor 1 and rIL-5 are responsible for the activity to partially induce CL-3 cells into IgM-synthesizing cells and also to synergize with IL-2 to augment IL-2R expression on and IgM synthesis in CL-3 cells. These actions of rIL-5 on a homogeneous cloned line (BCL1-CL-3 cells) allow us to identify and characterize the two alternated B cell developmental pathways. One is an IL-2-independent, IL-5-driven differentiation pathway without preceding up-regulated IL-2R expression, and the other is an IL-5 plus IL-2-dependent augmented differentiation pathway with preceding up-regulated IL-2R expression. We have also demonstrated the functional difference of two distinct B cell growth-promoting factors, B cell-stimulating factor 1 (rIL-4) and rIL-5. CL-3 cells are equally stimulated to grow by rIL-4 and rIL-5, whereas only rIL-5 can render CL-3 cells responsive to rIL-2, indicating that these two lymphokines affect B cells in a strikingly different manner.