IL-1beta suppresses prolonged Akt activation and expression of E2F-1 and cyclin A in breast cancer cells

Cell cycle aberrations occurring at the G(1)/S checkpoint often lead to uncontrolled cell proliferation and tumor growth. We recently demonstrated that IL-1beta inhibits insulin-like growth factor (IGF)-I-induced cell proliferation by preventing cells from entering the S phase of the cell cycle, leading to G(0)/G(1) arrest. Notably, IL-1beta suppresses the ability of the IGF-I receptor tyrosine kinase to phosphorylate its major docking protein, insulin receptor substrate-1, in MCF-7 breast carcinoma cells. In this study, we extend this juxtamembrane cross-talk between cytokine and growth factor receptors to downstream cell cycle machinery. IL-1beta reduces the ability of IGF-I to activate Cdk2 and to induce E2F-1, cyclin A, and cyclin A-dependent phosphorylation of a retinoblastoma tumor suppressor substrate. Long-term activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, but not the mammalian target of rapamycin or mitogen-activated protein kinase pathways, is required for IGF-I to hyperphosphorylate retinoblastoma and to cause accumulation of E2F-1 and cyclin A. In the absence of IGF-I to induce Akt activation and cell cycle progression, IL-1beta has no effect. IL-1beta induces p21(Cip1/Waf1), which may contribute to its inhibition of IGF-I-activated Cdk2. Collectively, these data establish a novel mechanism by which prolonged Akt phosphorylation serves as a convergent target for both IGF-I and IL-1beta; stimulation by growth factors such as IGF-I promotes G(1)-S phase progression, whereas IL-1beta antagonizes IGF-I-induced Akt phosphorylation to induce cytostasis. In this manner, Akt serves as a critical bridge that links proximal receptor signaling events to more distal cell cycle machinery.     

Contribution of IL-6 to the antiproliferative effect of IL-1 and tumor necrosis factor on tumor cell lines

The role of IL-6 in the antiproliferative effect of IL-1 for tumor cell lines was investigated using IL-1-sensitive cell lines. Human recombinant IL-1 alpha and IL-6 both inhibited the growth of an IL-1-sensitive cloned human melanoma cell line (A375-C6). However, IL-1 has greater maximum growth inhibitory activity than IL-6. Conditioned medium of the tumor cells that were treated with IL-1 contained IL-6 as determined by ELISA. Northern blot analysis revealed that IL-6 mRNA expression increased in IL-1-treated cells. In addition, antibody against human IL-6 neutralized about 50% of the antiproliferative effect of IL-1. The growth of an IL-1-resistant clone of A375 cells (A375-C5), which cannot be shown to express any detectable IL-1R, was inhibited by IL-6 to the same degree as A375-C6 cells. The A375-C5 cell line did not produce IL-6 or increase IL-6 mRNA after stimulation with IL-1. These results indicate that IL-6 mediates in part the antiproliferative effect of IL-1 on A375-C6 cells by acting as an autocrine antiproliferative factor. IL-1 also inhibited the growth of a malignant human mammary cell line (MDA-MB-415). IL-6 exhibited only slight growth inhibition in this cell line. Neither IL-6 production nor IL-6 mRNA expression was induced in this cell line by IL-1. Antibody against IL-6 did not neutralize the antiproliferative effect of IL-1. Therefore, for MDA-MB-415 cells IL-6 appeared not to be involved in the antiproliferative effect of IL-1. These results indicate that the antiproliferative effect of IL-1 involves at least two pathways, one IL-6 dependent and another IL-6 independent. The contribution of IL-6 to the antiproliferative effect of TNF was also examined. IL-6 appeared not to play a role in the antiproliferative effect of TNF in these cell lines.     

IL-7 splicing variant IL-7δ5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7δ5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The results showed that IL-7δ5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27(kip1) expression. Mechanistically, we found that IL-7δ5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7δ5. In conclusion, our findings demonstrate that IL-7δ5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7δ5 may be a potential target for human breast cancer therapeutics intervention.     

Role of ornithine decarboxylase in the regulation of cell growth by IL-1 and tumor necrosis factor

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine synthesis, and polyamines are required for cell growth. As an approach to clarifying the mechanism of action IL-1, the effects of IL-1 on ODC activity were examined in various cell lines whose proliferation was either suppressed or enhanced by IL-1. The proliferation of all cell types used in these experiments was markedly suppressed by a specific ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), substantiating the crucial role of ODC activity for cell proliferation. ODC activity also was considerably suppressed by IL-1 in those cells on which IL-1 exerts an antiproliferative effect, such as a human melanoma cell line (A375) and malignant human mammary cell lines (MCF-7 and T-47D). On the other hand, ODC activity was stimulated in cells that are stimulated to proliferate in response to IL-1, such as a mouse helper T cell line (D10.G4.1), a NK cell-like cell line (YT), and a human glioblastoma cell line (U373 MG). The effect of IL-1 on ODC activity preceded and directly correlated in a dose-dependent manner with its effect on DNA synthesis. Furthermore, putrescine, a product of the ODC reaction and a precursor of polyamines, was able to overcome most, but not all, the antiproliferative action of IL-1 in A375 melanoma cells, which were the most sensitive to suppression by IL-1. However, putrescine did not reverse the cytostatic effect of IL-1 on MCF-7 and T-47D cell lines. In contrast, putrescine, like IL-1, exhibited some co-mitogenic activity on D10.G4.1 cells. Because the biological activities of TNF and IL-1 show considerable overlap, the effect of TNF on ODC activity also was examined. TNF had an antiproliferative effect on A375 cells and stimulated the proliferation of U373 MG cells. The ODC activity in A375 cells was suppressed by TNF, and the ODC activity in U373 MG cells was stimulated by TNF. Putrescine also partially overcame the inhibitory effect of TNF. These results suggest that the regulation of ODC activity may be a key component in the antiproliferative and proliferative action of IL-1 and TNF in some tumor cell types.     

Relationship between cytokine-dependent cell cycle progression and MHC class II antigen expression by human CD34+ HLA-DR- bone marrow cells.

Human CD34+ HLA-DR- bone marrow cells constitute a phenotypically homogeneous population of quiescent cells. More than 97% of CD34+ HLA-DR- cells reside in the G0/G1 phase of the cell cycle. The in vitro effects of two cytokines, IL-1 alpha and IL-3, alone or in combination, on the viability, cell cycle status and acquisition of HLA-DR by this cell population were examined. Cell viability was preserved in cultures receiving cytokines, but declined steadily in cultures deprived of exogenous IL. Over a period of 4 days, IL-3 progressively induced the expression of HLA-DR although driving corresponding numbers of cells into S and G2 + M. Although IL-1 alpha induced the expression of HLA-DR, it was not as effective as IL-3 in promoting the exit of these cells from G0/G1. Combinations of IL-1 alpha and IL-3, however, exerted an even greater effect on promoting both HLA-DR expression and entry of cells into active phases of the cell cycle. Simultaneous measurement of HLA-DR expression and cell cycle status in response to IL-1 alpha and IL-3 indicated that the majority of de novo expression of HLA-DR occurred in cells that remained in G0/G1. CD34+ HLA-DR- cells cultured with IL-1 alpha and IL-3 but arrested in G0/G1 by hydroxyurea were still capable of expressing HLA-DR, demonstrating that the acquisition of HLA-DR was independent of the entry of these cells into active phases of the cell cycle. These data indicate that the survival, HLA-DR expression, and cell cycle status of human CD34+ HLA-DR- bone marrow cells are governed by regulatory cytokines such as IL-1 alpha and IL-3. In addition, the entry of these cells into active phases of the cell cycle does not seem to be a prerequisite for the expression of HLA-DR, nor does it seem that the acquisition of HLA-DR by hematopoietic progenitor cells is a marker of cells entering the S phase of the cell cycle.     

IL-4 inhibits cell cycle progression of human umbilical vein endothelial cells by affecting p53, p21(Waf1), cyclin D1, and cyclin E expression

IL-4 is emerging as a candidate cytokine for the treatment of inflammatory and autoimmune diseases. We have reported that IL-4 has anti-angiogenic activity and inhibits the growth of human umbilical vein endothelial cells (HUVEC) in response to vascular endothelial growth factor (VEGF) or fibroblast growth factor-2 (FGF-2). Cell cycle analysis of this effect revealed that IL-4 arrests the growth of FGF-2-stimulated HUVEC in G0 + G1 phases. The absence of subdiploid cells showed that it did not induce apoptosis. Growth arrest was dose-dependent, but the percentage of G0 + G1 phase cells never exceeded 85%. An immunoblot analysis demonstrated that expression of p53 and p21(Waf1) was increased and that of cyclin D1 and cyclin E decreased by IL-4. These results show that IL-4 inhibits endothelial cell growth by altering the expression of cell cycle regulatory molecules.     

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.     

Differentiation of HL-60 myeloid leukaemia cells is associated with a transient block in the G2 phase of the cell cycle

The human promyelocytic leukaemia cell line HL-60 can be induced to differentiate towards mature granulocytes by treatment with dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP). Differentiation begins within 16-24 h of treatment and is associated with a time- and dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle with a concomitant decrease in the number of cells in the S and G2 + M phases. Using acridine orange staining, we found that the RNA content of the cells also decreased following differentiation. Stathmokinetic analysis of HL-60 cell populations following dbcAMP treatment showed no effect on the total number of cells in the G0/G1 or S phases, or the rate of progression of cells through these cell cycle compartments. In contrast, dbcAMP was found to induce a transient arrest of the cells in the G2 phase. We also found that differentiation induced by dbcAMP did not require progression of the cells through the cell cycle. Cells arrested in either G1/S by hydroxyurea or G2 + M by colcemid eventually expressed markers of mature granulocytes. These results demonstrate that dbcAMP modulates cell cycle progression. However, these cell cycle changes alone are insufficient to induce granulocytic differentiation of HL-60 cells.     

Carbon monoxide produced by heme oxygenase-1 suppresses T cell proliferation via inhibition of IL-2 production

Heme oxygenase-1 (HO-1) catabolizes heme into CO, biliverdin, and free iron and serves as a protective enzyme by virtue of its anti-inflammatory, antiapoptotic, and antiproliferative actions. Previously, we have demonstrated that human CD4(+) T cells express HO-1 and that HO-1-overexpressing Jurkat T cells tend to display lower proliferative response. The aim of this study is to elucidate the mechanism(s) by which HO-1 can mediate its antiproliferative effect on CD4(+) T cells. Among the three HO-1 byproducts, only CO showed suppressive effect on T cell proliferation in response to anti-CD3 plus anti-CD28 Abs, mimicking the antiproliferative action of HO-1. CO blocked the cell cycle entry of T cells, which was independent of the guanylate cyclase/cGMP pathway. CO also suppressed the secretion of IL-2, and this suppressive effect of CO on IL-2 secretion mediated the antiproliferative action of CO. CO selectively inhibited the extracellular signal-regulated kinase pathway, which could explain the suppressive effects of CO on T cell proliferation and IL-2 secretion. Based on these findings, we suggest that HO-1/CO suppresses T cell proliferation and IL-2 secretion, possibly via its inhibition of extracellular signal-regulated kinase activation.     

Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.     

IL-22-mediated tumor growth reduction correlates with inhibition of ERK1/2 and AKT phosphorylation and induction of cell cycle arrest in the G2-M phase

IL-22 is a recently discovered cytokine of the IL-10 family that binds to a class II cytokine receptor composed of IL-22R1 and IL-10R2(c) and influences a variety of immune reactions. As IL-22 has also been shown to modulate cell cycle and proliferation mediators such as ERK1/2 and JNK, we studied the role of IL-22 in proliferation, apoptosis, and cell cycle regulation in EMT6 murine breast cancer cells in vitro and in vivo. In this study, we report that murine breast cancer cells express functional IL-22R as indicated by RT-PCR studies, immunoblotting, and STAT3 activation assays. Importantly, IL-22 exposure of EMT6 cells resulted in decreased levels of phosphorylated ERK1/2 and AKT protein kinases, indicating an inhibitory effect of IL-22 on signaling pathways promoting cell proliferation. Furthermore, IL-22 induced a cell cycle arrest of EMT6 cells in the G(2)-M phase. IL-22 reduced EMT6 cell numbers and the proliferation rate by approximately 50% as measured by [(3)H]thymidine incorporation. IL-22 treatment of EMT6 tumor-bearing mice lead to a decreased tumor size and a reduced tumor cell proliferation in vivo, as determined by 3'-deoxy-3'-fluorothymidine-positron emission tomography scans. Interestingly, IL-22 did not induce apoptosis, as determined in annexin V binding assay and caspase-3 activation assay and had no effect on angiogenesis in vivo. In conclusion, our results indicate that IL-22 reduced tumor growth by inhibiting signaling pathways such as ERK1/2 and AKT phosphorylation that promote tumor cell proliferation in EMT6 cells. Therefore, IL-22 may play a role in the control of tumor growth and tumor progression.     

IL-2 and IL-5 both induce mu S and J chain mRNA in a clonal B cell line, but differ in their cell-cycle dependency for optimal signaling.

We have found that a neoplastic Lyl+ B cell clone (BCL1-3B3) can be stimulated to secrete IgM by a Th1-derived cytokine, IL-2, and/or by a Th2-derived cytokine, IL-5. At suboptimal concentrations these interleukins acted synergistically to enhance IgM secretion. Both IL-2 and IL-5 induced increases in microseconds and J chain mRNA levels. In the presence of both ILs, increases in microseconds and J chain mRNA were additive and paralleled increases in IgM secretion. Using cells synchronized at the G1/S border with excess thymidine or in early G1 using isoleucine-deficient media, IL-2 and IL-5 differed in their cell-cycle dependency for signal transmission. IL-5 appeared to act preferentially in late G1 of the cell cycle. In contrast, IL-2 stimulated S and G2 phase cells slightly more efficiently than cells in G1 of the cell cycle. Furthermore, a twofold increase in high-affinity IL-2R was observed as the cells entered S phase. The results suggest that although IL-2 and IL-5 can independently and additively induce differentiation of the Lyl+ BCL1-3B3 cells, they differ in their point of action during the cell cycle.     

The RASSF1A tumor suppressor restrains anaphase-promoting complex/cyclosome activity during the G1/S phase transition to promote cell cycle progression in human epithelial cells

Multiple molecular lesions in human cancers directly collaborate to deregulate proliferation and suppress apoptosis to promote tumorigenesis. The candidate tumor suppressor RASSF1A is commonly inactivated in a broad spectrum of human tumors and has been implicated as a pivotal gatekeeper of cell cycle progression. However, a mechanistic account of the role of RASSF1A gene inactivation in tumor initiation is lacking. Here we have employed loss-of-function analysis in human epithelial cells for a detailed investigation of the contribution of RASSF1 to cell cycle progression. We found that RASSF1A has dual opposing regulatory connections to G(1)/S phase cell cycle transit. RASSF1A associates with the Ewing sarcoma breakpoint protein, EWS, to limit accumulation of cyclin D1 and restrict exit from G(1). Surprisingly, we found that RASSF1A is also required to restrict SCF(betaTrCP) activity to allow G/S phase transition. This restriction is required for accumulation of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 and the concomitant block of APC/C-dependent cyclin A turnover. The consequence of this relationship is inhibition of cell cycle progression in normal epithelial cells upon RASSF1A depletion despite elevated cyclin D1 concentrations. Progression to tumorigenicity upon RASSF1A gene inactivation should therefore require collaborating genetic aberrations that bypass the consequences of impaired APC/C regulation at the G(1)/S phase cell cycle transition.     

Binding of IL-1 beta to IL-1R type II at single cell level.

To gain information on the possible biologic role of IL-1R type II (IL-1RII), expression of the 68-kDa IL-1 binding protein on human lymphoblastoid B cells was investigated at single cell level. Binding of iodinated IL-1 beta was evaluated by autoradiography on cytosmears of IL-1RII positive B cell lines RAJI, the RAJI clone 1H7, and STS 25. Results obtained suggest an heterogeneity of IL-1RII expression within the B cell population, with only 5 to 16% of the cells able to bind IL-1 beta. Up-regulation of IL-1RII expression by dexamethasone, evident in conventional binding assays, was achieved through both increase in the number of IL-1 binding cells (14-30%) and augmentation of receptor density on positive cells, By combining autoradiography with immunocytochemical staining, it could be shown that about 80% of IL-1RII + cells were negative for Ki67, a nuclear antigen expressed from late G1 to M phase. Cell cycle dependent expression of IL-1RII was confirmed on cells enriched in different phases of the cell cycle by counterflow centrifugal elutriation. It is thus proposed that IL-1RII is associated to the cell cycle.     

Paeoniflorin inhibits cell growth and induces cell cycle arrest through inhibition of FoxM1 in colorectal cancer cells

Paeoniflorin (PF) exhibits tumor suppressive functions in a variety of human cancers. However, the function of PF and molecular mechanism in colorectal cancer are elusive. In the present study, we investigated whether PF could exert its antiproliferative activity, anti-migration, and anti-invasive function in colorectal cancer cells. We found that PF inhibited cell growth and induced apoptosis and blocked cell cycle progression in the G0/G1 phase in colorectal cancer cells. Moreover, we found that PF suppressed cell migration and invasion in colorectal cancer cells. FoxM1 has been reported to play an important oncogenic role in human cancers. We also determine whether PF inhibited the expression of FoxM1, leading to its anti-cancer activity. We found that PF treatment in colorectal cancer cells resulted in down-regulation of FoxM1. The rescue experiments showed that overexpression of FoxM1 abrogated the tumor suppressive function induced by PF treatment. Notably, depletion of FoxM1 promoted the anti-tumor activity of PF in colorectal cancer cells. Therefore, inhibition of FoxM1 could participate in the anti-tumor activity of PF in colorectal cancer cells.     

Interleukin-1 modulation of human placental trophoblast proliferation

During early pregnancy, interleukin-1 (IL-1) is mainly produced and secreted by maternal decidua. Yet, its biological function on placental cells is not well defined. In this study, we employed JAR choriocarcinoma cell line as a model of human placental trophoblast to study the effect of IL-1. Treatment with recombinant human IL-1beta resulted in significant inhibition of JAR proliferation (P < .05) paralleled with increased cytotoxicity. The inhibitory effect was blocked by both IL-1 receptor antagonist (IL-1Ra) and antihuman IL-1beta monoclonal antibody. Analyzing the mode of action, IL-1beta was found to induce cell cycle arrest in the G0/G1 phase and triggered apoptotic cell death. These findings demonstrated that IL-1 regulates human trophoblast growth by induction of cell cycle delay and cell death.     

Enhanced Sensitivity of G1 Arrested Human Cancer Cells Suggests a Novel Therapeutic Strategy Using a Combination of Simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.

Key Words

TRAIL, Synchronization, Simvastatin, Cancer Therapy, Lovastatin, Cell Cycle, Apoptosis     

Comparative effects of tumor necrosis factor-alpha and IL-1 beta on mitogen-induced T cell activation

The effect of rTNF-alpha on human T cell function was examined and compared with that of rIL-1 beta by assessing the ability of each cytokine to support mitogen-induced proliferation, IL-2 production, and IL-2R expression. TNF-alpha and IL-1 beta each enhanced DNA synthesis induced by PHA or immobilized mAb to the CD3 molecular complex. In addition, each cytokine increased the number of cells entering the G1 phase of the cell cycle and augmented IL-2R expression. The combination of optimal concentrations of these factors supported these responses to a greater extent than either cytokine alone, suggesting that T cell responsiveness is independently regulated by the action of at least two separate monocyte derived cytokines. Whereas TNF-alpha had little effect, IL-1 beta augmented IL-2 mRNA expression and IL-2 production by mitogen-stimulated cells. Furthermore, IL-1 beta enhanced proliferation with increasing length of culture. Whereas TNF-alpha also enhanced proliferation late in culture, it was less effective in this regard than IL-1 beta. Thus, IL-1 beta and TNF-alpha augment mitogen-induced T cell proliferation by increasing the number of cells initially activated and by promoting subsequent cell cycle progression. They differ, however, in their capacity to promote IL-2 mRNA and IL-2 production and therefore ongoing T cell proliferation.     

Interleukin 5 (IL-5) can act in G0/G1 to induce S phase entry in B lymphocytes from normal and autoimmune strain mice and in transformed leukemic B cells

In addition to its ability to enhance antibody secretion, Interleukin 5 (IL-5) enhances murine B lymphocyte proliferation. This so-called growth factor activity has been amply demonstrated by many laboratories assessing thymidine incorporation or cell recovery. Attempts to actually quantitate the fraction of fresh splenic B cells responding to IL-5, by limiting dilution analysis or other means, with few exceptions have yielded disappointingly small numbers--generally between 1 and 5%, or perhaps less. We have recently identified the peritoneal cavity as a reservoir rich in IL-5-responsive B cells. In this report, we provide independent corroboration of this high IL-5 reactivity by means of cell cycle analysis. Low-density peritoneal B cells, more than 90% of which are in G0 and G1 phases, were stimulated with polyclonal activators in the presence of mitotic inhibitors. Frequencies of IL-5-responsive B cells were measured by observing the differences in the proportions of cultured cells entering S and later phases in the presence, compared to the absence, of IL-5. Some 10 to 20% more of the low-density peritoneal B cells from normal mice entered S phase when IL-5 was present with LPS + DXS. A similar IL-5-mediated elevation in the frequency of S phase entry was seen with peritoneal B cells from the autoimmune mouse strain NZB. Furthermore, a measurable fraction of peritoneal B cells from these mice were even capable of responding to IL-5 alone. These IL-5-induced increases could be blocked by anti-IL-5 mAb. About 30% of the BCL1 leukemic B cell line initiated DNA replication when stimulated with IL-5 alone. Hence, IL-5-responsive B cell fractions have been measured for some normal, autoimmune strain and transformed leukemic B cell phenotypes. In addition to quantitating the proportion of IL-5-responsive B cells, these experiments formally demonstrate that IL-5 can act in the G1 phase to increase S phase entry.