GM-CSF, IL-3, and IL-5 Family of Cytokines: Regulators of Inflammation.

The ß common chain cytokines GM-CSF, IL-3, and IL-5 regulate varied inflammatory responses that promote the rapid clearance of pathogens but also contribute to pathology in chronic inflammation. Therapeutic interventions manipulating these cytokines are approved for use in some cancers as well as allergic and autoimmune disease, and others show promising early clinical activity. These approaches are based on our understanding of the inflammatory roles of these cytokines; however, GM-CSF also participates in the resolution of inflammation, and IL-3 and IL-5 may also have such properties. Here, we review the functions of the ß common cytokines in health and disease. We discuss preclinical and clinical data, highlighting the potential inherent in targeting these cytokine pathways, the limitations, and the important gaps in understanding of the basic biology of this cytokine family.

In the absence of apoptosis, myeloid cells arrest when deprived of growth factor, but remain viable by consuming extracellular glucose.

Withdrawal of the growth factor interleukin-3 (IL-3) from IL-3-dependent myeloid cells causes them to undergo Bax/Bak1-dependent apoptosis, whereas factor-deprived Bax-/-Bak1-/- cells remain viable, but arrest and shrink. It was reported that withdrawal of IL-3 from Bax-/-Bak1-/- cells caused decreased expression of the glucose transporter Glut1, leading to reduced glucose uptake, so that arrested cells required Atg5-dependent autophagy for long-term survival. In other cell types, a decrease in Glut1 is mediated by the thioredoxin-interacting protein (Txnip), which is induced in IL-3-dependent myeloid cells when growth factor is removed. We mutated Atg5 and Txnip by CRISPR/Cas9 and found that Atg5-dependent autophagy was not necessary for the long-term viability of cycling or arrested Bax-/-Bak1-/- cells, and that Txnip was not required for the decrease in Glut1 expression in response to IL-3 withdrawal. Surprisingly, Atg5-deficient Bax/Bak1 double mutant cells survived for several weeks in medium supplemented with 10% fetal bovine serum (FBS), without high concentrations of added glucose or glutamine. When serum was withdrawn, the provision of an equivalent amount of glucose present in 10% FBS (~0.5 mM) was sufficient to support cell survival for more than a week, in the presence or absence of IL-3. Thus, Bax-/-Bak1-/- myeloid cells deprived of growth factor consume extracellular glucose to maintain long-term viability, without a requirement for Atg5-dependent autophagy.

Interleukin-3 stimulates matrix metalloproteinase 12 production from macrophages promoting thoracic aortic aneurysm/dissection.

Thoracic aortic aneurysm and dissection (TAAD) is due to degeneration of the aorta and causes a high mortality rate, while molecular mechanisms for the development of TAAD are still not completely understood. In the present study, 3-aminopropionitrile (BAPN) treatment was used to induce TAAD mouse model. Through transcriptome analysis, we found the expression levels of genes associated with interleukin-3 (IL-3) signaling pathway were up-regulated during TAAD development in mouse, which were validated by real-time PCR. IL-3 positive cells were increased in TAAD mouse aortas, especially for smooth muscle cells (SMCs). IL-3 deficiency reduced BAPN-induced TAAD formation. We then examined the matrix metalloproteinases (MMPs) expression during TAAD formation in both wild-type and IL-3 deficient mice, showing that MMP12 were significantly down-regulated in IL-3 deficient aortas. Mechanistically, we found recombinant IL-3 could increase MMP12 production and activity from macrophages in vitro Silencing of IL-3 receptor ß, which was mainly expressed in macrophages but not SMCs, diminished the activation of c-Jun N terminal kinase (JNK)/extracellular-regulated protein kinases 1/2 (ERK1/2)/AP-1 signals, and decreased MMP12 expression in IL-3 stimulated macrophages. Moreover, both circulating and aortic inflammation were decreased in IL-3 deficient aortas. Taken together, our results demonstrated that IL-3 stimulated the production of MMP12 from macrophages by a JNK- and ERK1/2-dependent AP-1 pathway, contributing to TAAD formation. Thus, the IL-3/IL-3Rß/MMP12 signals activation may be an important pathological mechanism for progression of TAAD.

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Biophysical changes reduce energetic demand in growth factor-deprived lymphocytes.

Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-xL expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-xL cells depleted of IL-3 and primary CD8(+) T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress.

Interleukin-3-deficient mice have increased resistance to blood-stage malaria.

The contribution of interleukin-3 (IL-3), a hematopoietic growth factor and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting IL-3-deficient (knockout [KO]) mice with Plasmodium berghei NK65. Male IL-3 KO mice, but not female mice, were more resistant to infection than wild-type (WT) mice, as evidenced by lower peak parasitemia and prolonged survival. Both male and female IL-3 KO mice had increased splenomegaly and were more anemic than corresponding WT mice. Anemia was compensated for by an increase in bone marrow and splenic erythropoiesis in IL-3 KO mice, as evidenced by higher levels of erythroid progenitors. Plasma levels of gamma interferon (IFN-γ) and CXCL9 (monokine induced by IFN-γ [MIG]) were found to be significantly reduced in IL-3 KO mice during early stages of infection. In contrast, granulocyte colony-stimulating factor (G-CSF) levels were significantly higher, and the percentage of peripheral blood neutrophils lower, in infected IL-3 KO mice than in WT counterparts. Overall, our results indicate that IL-3 plays a critical role in suppressing protective immunity to P. berghei NK65 infection and that it is involved in inhibiting the development of splenomegaly, anemia, and erythropoiesis. IL-3 also influences IFN-γ, CXCL9, and G-CSF production in response to infection. The abnormal responses seen in infected IL-3 KO mice may be due to the lack of IL-3 during development, to the lack of IL-3 in the infected mature mice, or to both.

Trib3 is regulated by IL-3 and affects bone marrow-derived mast cell survival and function.

Mast cells are the principal effectors of IgE-mediated immune responses, including allergic reactions. Tribbles homolog 3 (Trib3) encodes a pseudokinase implicated in the cellular stress response and has been linked to inflammation in certain situations. Here we report the role of Trib3 in mouse bone marrow-derived mast cells (BMMCs). Our results show that Trib3 mRNA expression in BMMCs is positively regulated by the growth factor interleukin (IL)-3. BMMCs originating from Trib3 knockout mice demonstrate unaltered differentiation kinetics and cell surface expression of mast cell markers. When challenged with transient IL-3 deprivation, Trib3-deficient BMMCs display delayed recovery, and during prolonged IL-3 starvation, cell death is accelerated in Trib3-null cultures. IgE-dependent and pharmacologically induced degranulation is impaired in Trib3-deficient BMMCs, as is activation-induced cytokine mRNA expression. Thus, Trib3 contributes to the survival and activity of primary cultured mast cells, which suggests a role for Trib3 in the modulation of the immune response.

ATP release from dying autophagic cells and their phagocytosis are crucial for inflammasome activation in macrophages.

Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1ß while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1ß secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P(2)X(7) purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses.

A dual role for the immune response in a mouse model of inflammation-associated lung cancer.

Lung cancer is the leading cause of cancer death worldwide. Both principal factors known to cause lung cancer, cigarette smoke and asbestos, induce pulmonary inflammation, and pulmonary inflammation has recently been implicated in several murine models of lung cancer. To further investigate the role of inflammation in the development of lung cancer, we generated mice with combined loss of IFN-γ and the ß-common cytokines GM-CSF and IL-3. These immunodeficient mice develop chronic pulmonary inflammation and lung tumors at a high frequency. Examination of the relationship between these tumors and their inflammatory microenvironment revealed a dual role for the immune system in tumor development. The inflammatory cytokine IL-6 promoted optimal tumor growth, yet wild-type mice rejected transplanted tumors through the induction of adaptive immunity. These findings suggest a model whereby cytokine deficiency leads to oncogenic inflammation that combines with defective antitumor immunity to promote lung tumor formation, representing a unique system for studying the role of the immune system in lung tumor development.

Cutting edge: basophils are transiently recruited into the draining lymph nodes during helminth infection via IL-3, but infection-induced Th2 immunity can develop without basophil lymph node recruitment or IL-3.

Basophils are recognized as immune modulators through their ability to produce IL-4, a key cytokine required for Th2 immunity. It has also recently been reported that basophils are transiently recruited into the draining lymph node (LN) after allergen immunization and that the recruited basophils promote the differentiation of naive CD4 T cells into Th2 effector cells. Using IL-3(-/-) and IL-3Rbeta(-/-) mice, we report in this study that the IL-3/IL-3R system is absolutely required to recruit circulating basophils into the draining LN following helminth infection. Unexpectedly, the absence of IL-3 or of basophil LN recruitment played little role in helminth-induced Th2 immune responses. Moreover, basophil depletion in infected mice did not diminish the development of IL-4-producing CD4 T cells. Our results reveal a previously unknown role of IL-3 in recruiting basophils to the LN and demonstrate that basophils are not necessarily associated with the development of Th2 immunity during parasite infection.

Role of GM-CSF signaling in cell-based tumor immunization.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent adjuvant in cancer vaccination; however, the specific role of endogenous GM-CSF remains unknown. We performed cell-based vaccination in 2 tumor models. First, we vaccinated C57BL/6 mice lacking either GM-CSF, IL-5, or beta-common chain (betac), a receptor subunit essential for GM-CSF and IL-5 signaling, with melanoma cells engineered to produce GM-CSF. Tumor vaccination was effective in both GM-CSF(-/-) and IL-5(-/-) mice, showing that protective immunization is independent of both endogenous cytokines. However, all betac(-/-) animals developed tumor. Loss of tumor immunity in betac(-/-) mice does not reflect global impairment in cell-mediated immunity, as contact hypersensitivity reaction to haptens is unaltered. The importance of tumor cell-derived GM-CSF was highlighted by recruitment of dendritic cells at the vaccination site in wild-type, GM-CSF(-/-), and IL-5(-/-) but not in betac(-/-) mice. In the second model, vaccination with unmodified RENCA cells showed similar results with efficient immunization in BALB/c wild-type and GM-CSF(-/-), whereas all betac(-/-) animals died. Altogether, our results strongly suggest that although endogenous GM-CSF and IL-5 are not required to induce tumor immunity, signaling through betac receptor is critically needed for efficient cancer vaccination in both genetically modified GM-CSF-secreting tumor cells and a spontaneously immunogenic models.

IL-3 induces basophil expansion in vivo by directing granulocyte-monocyte progenitors to differentiate into basophil lineage-restricted progenitors in the bone marrow and by increasing the number of basophil/mast cell progenitors in the spleen.

Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in approximately 4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3R alpha-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen.

Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim.

Bcl-2 family members regulate apoptosis in response to cytokine withdrawal and a broad range of cytotoxic stimuli. Pro-apoptotic Bcl-2 family members Bax and Bak are essential for apoptosis triggered by interleukin-3 (IL-3) withdrawal in myeloid cells. The BH3-only protein Puma is critical for initiation of IL-3 withdrawal-induced apoptosis, because IL-3-deprived Puma(-/-) cells show increased capacity to form colonies when IL-3 is restored. To investigate the mechanisms of Puma-induced apoptosis and the interactions between Puma and other Bcl-2 family members, we expressed Puma under an inducible promoter in cells lacking one or more Bcl-2 family members. Puma rapidly induced apoptosis in cells lacking the BH3-only proteins, Bid and Bim. Puma expression resulted in activation of Bax, but Puma killing was not dependent on Bax or Bak alone as Puma readily induced apoptosis in cells lacking either of these proteins, but could not kill cells deficient for both. Puma co-immunoprecipitated with the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1 but not with Bax or Bak. These data indicate that Puma functions, in the context of induced overexpression or IL-3 deprivation, primarily by binding and inactivating anti-apoptotic Bcl-2 family members.

Functional deficiencies of granulocyte-macrophage colony stimulating factor and interleukin-3 contribute to insulitis and destruction of beta cells.

The pathogenesis of type 1 diabetes (T1D) involves the immune-mediated destruction of insulin-producing beta cells in the pancreatic islets of Langerhans. Genetic analysis of families with a high incidence of T1D and nonobese diabetic (NOD) mice, a prototypical model of the disorder, uncovered multiple susceptibility loci, although most of the underlying immune defects remain to be delineated. Here we report that aged mice doubly deficient in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) manifest insulitis, destruction of insulin-producing beta cells, and compromised glucose homeostasis. Macrophages from mutant mice produce increased levels of p40 after LPS stimulation, whereas concurrent ablation of interferon-gamma (IFN-gamma) ameliorates the disease. The administration of antibodies that block cytotoxic T lymphocyte associated antigen-4 (CTLA-4) to young mutant mice precipitates the onset of insulitis and hyperglycemia. These results, together with previous reports of impaired hematopoietic responses to GM-CSF and IL-3 in patients with T1D and in NOD mice, indicate that functional deficiencies of these cytokines contribute to diabetes.

Caspase inhibition blocks cell death and results in cell cycle arrest in cytokine-deprived hematopoietic cells.

Cytokine deprivation has been classically used to study molecular processes of apoptosis. Following interleukin (IL)-3 withdrawal in FL5.12 cells, Bax undergoes a conformational change that results in its mitochondria targeting, cytochrome c release, activation of caspase-9, and apoptosis. Cells overexpressing Casp9DN (dominant negative caspase-9) or treated with the caspase inhibitor Q-VD-OPh increased viability but failed to increase clonogenic survival. We find that caspase-inhibited cells had a significant fraction of viable cells (herein termed "rescued" cells) that failed to initiate cell division after IL-3 add back. The "rescued" cells had reduced mitochondrial potential, stained for active Bax, and had reduced staining with dihydroethidium, an agent sensitive to superoxide levels. Readdition of IL-3 after deprivation demonstrated that Bax activation was reversed, whereas altered 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and dihydroethidium staining persisted for days. Furthermore, the "rescued" cells were resistant to rotenone, an inhibitor of mitochondrial respiration. The cells were highly sensitive to 2-deoxyglucose, an inhibitor of glycolysis and proposed anti-cancer agent. We conclude that the inhibition of caspase-9 allows cells to retain viability, but cells have prolonged mitochondrial dysfunction and enter a unique nondividing state that shares some properties with malignant cells.

A constitutively active Lck kinase promotes cell proliferation and resistance to apoptosis through signal transducer and activator of transcription 5b activation.

Lck is a Src family protein tyrosine kinase and is expressed predominantly in T cells. Aberrant expression or activation of Lck kinase has been reported in both lymphoid and nonlymphoid malignancies. However, the mechanisms underlying Lck-mediated oncogenesis remain largely unclear. In this report, we establish a tetracycline-inducible system to study the biochemical and biological effects of a constitutively active Lck mutant with a point mutation at the negative regulatory tyrosine. Expression of the active Lck kinase induces both tyrosine phosphorylation and DNA-binding activity of signal transducer and activator of transcription 5b (STAT5b), a STAT family member activated in a variety of tumor cells. The active Lck kinase interacts with STAT5b in cells, suggesting that Lck may directly phosphorylate STAT5b. Expression of the constitutively active Lck mutant in interleukin-3 (IL-3)-dependent BaF3 cells promotes cell proliferation. In addition, the active Lck kinase protects BaF3 cells from IL-3 withdrawal-induced apoptotic death and leads to IL-3-independent growth. These transforming properties of the oncogenic Lck kinase can be further augmented by expression of exogenous wild-type STAT5b but attenuated by a dominant-negative form of STAT5b. All together, our results suggest the potential involvement of STAT5b in Lck-mediated cellular transformation.

Upregulation of alpha globin promotes apoptotic cell death in the hematopoietic cell line FL5.12.

The function of alpha globin in the context of oxygen transport in erythroid cells is well described. Recently the expression of alpha globin was shown to be upregulated upon specific apoptotic stimuli like cytokine deprivation or cisplatin treatment in the hematopoietic pro-B cell line, FL5.12. In contrast to alpha globin, beta globin or globin-like genes were expressed at a very low level or were not expressed at all. Further, we found that alpha globin was not associated with heme. Apoptotic cells neither produced hemoglobin nor displayed a phenotype of cells differentiating down the erythroid lineage. Also other cell lines of variable differentiation status (NIH3T3, HeLa, K562) upregulated alpha globin during treatment with apoptosis-inducing agents. Under IL-3-deprived conditions GFP-alpha globin accelerated the progression of apoptosis comparable to GFP-Bax. GFP-alpha globin was expressed at a low level and enrichment of FL5.12 cells expressing GFP-alpha globin was difficult even in the presence of IL-3. Caspase-8, -9 and -3 as well as the proapoptotic factor Bax and cytochrome c were activated. Antisense alpha globin downregulated the expression of endogenous alpha globin und reduced caspase activity. Taken together these data indicate that alpha globin is a new and crucial factor in apoptosis especially supporting the mitochondrial pathway.

Hematopoietic transcription factor GATA-2 promotes upregulation of alpha globin and cell death in FL5.12 cells.

Recently we showed that alpha globin is a novel pro-apoptotic factor in programmed cell death in the pro-B cell line, FL5.12. Alpha globin was also upregulated in various other cell lines after different apoptotic stimuli. Under withdrawal of IL-3, overexpression of alpha globin accelerated apoptosis in FL5.12. Here, we have studied how transcription of alpha globin is placed in the broader context of apoptosis. We used Affymetrix chip technology and RT QPCR to compare expression patterns of FL5.12 cells growing with or without IL-3 to search for transcription factors which were concomitantly upregulated with alpha globin. The erythroid-specific transcription factor GATA-2 was the earliest and most prominently upregulated candidate. GATA-1 was expressed at low levels and was weakly induced while GATA-3 was completely absent. To evaluate the influence of GATA-2 on alpha globin expression and cell viability we overexpressed GATA-2 in FL5.12 cells. Interestingly, high expression of GATA-2 resulted in cell death and elevated alpha globin levels in FL5.12 cells. Transduction of antisense GATA-2 prevented both increase of GATA-2 and alpha globin under apoptotic conditions and delayed cell death. We suggest a role of GATA-2 in apoptosis besides its function in maintenance and proliferation of immature hematopoietic progenitors.

The FLT3 internal tandem duplication mutation prevents apoptosis in interleukin-3-deprived BaF3 cells due to protein kinase A and ribosomal S6 kinase 1-mediated BAD phosphorylation at serine 112.

Internal tandem duplication (ITD) mutations in the FLT3 tyrosine kinase have been detected in approximately 20% of acute myeloid leukemia (AML) patients. Patients harboring FLT3/ITD mutations have a relatively poor prognosis. FLT3/ITD results in constitutive autophosphorylation of the receptor and factor-independent survival. Previous studies have shown that FLT3/ITD activates the signal transducers and activators of transcription 5 (STAT5), p42/p44 mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase (ERK) 1/2], and phosphatidylinositol 3-kinase/Akt pathways. We herein provide biochemical and biological evidence that ribosomal S6 kinase 1 (RSK1) and protein kinase A (PKA) are the two principal kinases that mediate the antiapoptotic function of FLT3/ITD via phosphorylation of BAD at Ser112. Inhibiting both MAPK kinase (MEK)/ERK and PKA pathways by a combination of U0126 (10 micromol/L) and H-89 (5 micromol/L) reduced most of BAD phosphorylation at Ser112 and induced apoptosis to a level comparable with that induced by FLT3 inhibitor AG1296 (5 micromol/L) in BaF3/FLT3/ITD cells. RNA interference of RSK1 or PKA catalytic subunit reduced BAD phosphorylation and induced apoptosis. The MEK inhibitor U0126 and/or the PKA inhibitor H-89 greatly enhanced the efficacy of the FLT3 inhibitor AG1296, suggesting that combining FLT3/ITD downstream pathway inhibition with FLT3 inhibitors may be a viable therapeutic strategy for AML caused by a FLT3/ITD mutation.

Extracellular ATP inhibits apoptosis and maintains cell viability by inducing autocrine production of interleukin-4 in a myeloid progenitor cell line.

Interleukin-3 (IL-3)-dependent myeloid progenitor cell FDC.P2 is induced to undergo apoptotic cell death upon IL-3 depletion. Extracellular adenosine triphosphate (ATP) was found to prevent apoptosis and maintain cell viability of FDC.P2 cells upon IL-3 withdrawal. The antiapoptotic effect of ATP required extracellular Ca2+. Furthermore, FK506, a specific inhibitor of calcium/calmodulin-dependent protein phosphatase calcineurin, inhibited the antiapoptotic effect of ATP. As one of cytokines whose expression is dependent on the activation of calcineurin, interleukin-4 (IL-4) played a critical role in ATP-mediated cell survival of FDC.P2 cells because neutralizing antibody against IL-4 effectively abrogated the antiapoptotic activity of ATP. Moreover, ATP treatment induced a significant amount of secreted IL-4 that was sufficient to maintain cell viability. Taken together, our present results demonstrate that extracellular ATP triggers autocrine production of IL-4 through calcium-dependent activation of calcineurin and secreted IL-4 substitutes IL-3 in protecting FDC.P2 cells from apoptosis even in the absence of IL-3.