Novel human neutrophil agonistic properties of arsenic trioxide: involvement of p38 mitogen-activated protein kinase and/or c-jun NH2-terminal MAPK but not extracellular signal-regulated kinases-1/2

Arsenic trioxide (ATO) is known for treating acute promyelocytic leukemia and for inducing apoptosis and mitogen-activated protein kinases (MAPKs) in promyelocytes and cancer cells. We recently reported that ATO induces neutrophil apoptosis. The aim of this study was to establish whether or not ATO recruits MAPKs in neutrophils, as well as to further investigate its agonistic properties. We found that ATO activates p38 and that, unlike H2O2, this response was not inhibited by exogenous catalase. Also, we demonstrated that ATO-induced p38 activation occurs before H2O2 generation and without a calcium burst. We next established that ATO recruits c-jun NH2-terminal (JNK) but not extracellular signal-regulated kinase 1 and 2 (Erk-1/2). Using pharmacological inhibitors, we found that the proapoptotic activity of ATO occurs by a MAPK-independent mechanism. In contrast, the ability of ATO to enhance adhesion, migration, phagocytosis, release, and activity of gelatinase and degranulation of secretory, specific, and gelatinase, but not azurophilic granules, is dependent upon activation of p38 and/or JNK. This is the first study establishing that ATO possesses important agonistic properties in human neutrophils. Given the central role of neutrophils in various inflammatory disorders, we propose that ATO might have broader therapeutic implications in clinics, especially for regulating inflammation.     

Possible pathophysiological roles of mitogen-activated protein kinases (MAPKs) in endometriosis

PROBLEM: Endometriosis accompanies local inflammatory reactions in the peritoneal cavity. We examined the phosphorylation of mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and c-Jun N-terminal kinase (JNK) in endometriotic stromal cells, and their possible pathophysiological roles in endometriosis in relation to proinflammatory substances. METHOD OF STUDY: Endometriotic stromal cells were isolated from endometriomas and were cultured for the experiments. Phosphorylation of MAPKs in endometriotic stromal cells treated with interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and H(2)O(2) were examined by Western blot analysis. Effects of PD98059, SB202190 and SP600125 (inhibitors of ERK, p38 and JNK, respectively) on IL-1beta-induced secretion of IL-6 and IL-8, and on IL-1beta-induced expression of cyclo-oxygenase-2 (COX-2) in endometriotic cells were studied. In addition, eutopic endometrial tissues were collected, and the phosphorylation rate of p38 in eutopic endometrial tissues and endometriotic tissues were determined. RESULTS: IL-1beta, TNFalpha and H(2)O(2) stimulated the phosphorylation of ERK, p38 and JNK, while the total amounts of proteins of the respective MAPKs were virtually the same compared with those in the unstimulated controls. Both SB202190 and SP600125 suppressed IL-1beta-induced secretion of IL-6 and IL-8, and PD98059 suppressed IL-1beta-induced secretion of IL-8. Both SB202190 and PD98059 suppressed IL-1beta-induced expression of COX-2 in endometriotic cells. The p38 phosphorylation rates in the endometriotic tissues were significantly higher than those in the eutopic endometrial tissues of the same patients. CONCLUSIONS: Given the current theory that inflammatory changes are involved in the progression of endometriosis, MAPKs could play as pivotal intracellular signal transducers in endometriotic cells, and thus have a pathophysiological role in the disease.     

c-Jun NH2-terminal kinase-mediated signaling is essential for Pseudomonas aeruginosa ExoS-induced apoptosis

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa mainly affects immunocompromised individuals as well as patients with cystic fibrosis. In a previous study, we showed that ExoS of P. aeruginosa, when injected into host cells through a type III secretion apparatus, functions as an effector molecule to trigger apoptosis in various tissue culture cells. Here, we show that injection of the ExoS into HeLa cells activates c-Jun NH(2)-terminal kinase (JNK) phosphorylation while shutting down ERK1/2 and p38 phosphorylation. Inhibiting JNK activation by expression of a dominant negative JNK1 or with a specific JNK inhibitor abolishes ExoS-triggered apoptosis, demonstrating the requirement for JNK-mediated signaling. Following JNK phosphorylation, cytochrome c is released into the cytosol, leading to the activation of caspase 9 and eventually caspase 3. Although c-Jun phosphorylation is also observed as a result of JNK activation, ongoing host protein synthesis is not essential for the apoptotic induction, suggesting that c-Jun- or other AP-1-driven activation of gene expression is dispensable in this process. Therefore, ExoS has opposing effects on different cellular pathways that regulate apoptosis: it shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2 leading ultimately to cytochrome c release and activation of caspases. These results highlight the modulation of host cell signaling by the type III secretion system during interaction between P. aeruginosa and host cells.     

The protective role of arjunolic acid against doxorubicin induced intracellular ROS dependent JNK-p38 and p53-mediated cardiac apoptosis

In spite of tremendous demand for the development and implementation of effective therapeutic strategies, limitations are still associated with doxorubicin-induced cardiotoxicity. Arjunolic acid (AA) has been shown to possess a multitude of biological functions. The purpose of the present study was to explore whether AA plays any protective role against doxorubicin-induced cardiotoxicity; and if so, what molecular mechanism it utilizes for its protective action. In rat cardiomyocytes, doxorubicin administration activated the proapoptotic p53, p38 and JNK MAPKs, Bax translocation, disrupted mitochondrial membrane potential, precipitated mitochondrion mediated caspase-dependent apoptotic signalling and reduced viability of cardiomyocytes. Doxorubicin exposure increases dichlorofluorescein (DCF) intensity corresponding to the intracellular H(2)O(2) generation in myocytes; catalase (CAT) treatment, however, reduced this intensity and preserves cell viability. Intracellular H(2)O(2) thus produced now activates the p38-JNK and p53-mediated pathways. CAT treatment also markedly decreased the doxorubicin-mediated activation of p38 and JNK, suggesting that H(2)O(2) is involved in the activation of MAPKs. Blockage of p53 and p38-JNK by pharmacological inhibitors also suppressed the doxorubicin-induced apoptosis with the concomitant inhibition of anti-apoptotic Bcl-2 family proteins. AA treatment ameliorates nearly all of these apoptotic actions of doxorubicin and preserves cell viability. Similarly, rats treated with doxorubicin displayed retarded growth of body and heart as well as elevated apoptotic indices in heart tissue, whereas AA treatment effectively neutralised all these doxorubicin-induced cardiac-abnormalities. Combining all, our results suggest that doxorubicin induces cardiac apoptosis via the activation of JNK-p38 and p53-mediated signalling pathways, where H(2)O(2) acts as the mediators of these pathways. AA can effectively and extensively counteract this action of doxorubicin, and may potentially protect the heart and cardiomyocytes from the severe doxorubicin-induced cardiovascular burden.     

Minocycline protects melanocytes against H2O2-induced cell death via JNK and p38 MAPK pathways

Vitiligo is an acquired and progressive disorder manifested by the selective destruction of melanocytes in the skin. An extremely high level of hydrogen peroxide (H2O2) in plasma as well as in lesional skin has been reported in vitiligo patients. High H2O2 level has been suggested to be responsible for the disappearance of melanocytes in vitiligo. JNK and p38 MAPK are strongly induced by oxidative stress and related to neuron loss in neurodegenerative disorders. Minocycline, an antibiotic possessing antioxidant activity, is capable of attenuating oxidative stress-induced neurotoxicity. To investigate whether minocycline rescues melanocytes from H2O2-induced apoptosis, cultured mouse melanocytes (B10BR) were treated with H2O2 in the presence or absence of minocycline. Our data showed that H2O2 decreases cell viability in a concentration-dependent manner which is attenuated by minocycline. Also, H2O2 treatment activates JNK and p38 MAPK, and executive caspase 3 in B10BR cells. Minocycline significantly inhibits H2O2-induced activation of JNK, p38 MAPK and caspase 3. Collectively, we concluded that minocycline protects melanocytes against H2O2-induced apoptosis in vitro. Its protective effect is associated with the inhibition of JNK and p38 MAPK. Our findings suggest that minocycline, a clinically well-tolerated, safe antibiotic, may be used to prevent melanocyte loss in the early stage of vitiligo.     

NGF protects Dorsal Root Ganglion neurons from oxaliplatin by modulating JNK/Sapk and ERK1/2

The involvement of the Mitogen-Activated Protein Kinases (MAPKs) family in platinum derivative-induced peripheral neuropathy has already been demonstrated. In particular, it has been evidenced that in Dorsal Root Ganglion (DRG) neurons prolonged exposure to oxaliplatin (OHP) induces early activation of p38 and ERK1/2, which mediate neuronal apoptosis, while the neuroprotective action of JNK/Sapk is downregulated by the drug treatment. In this study, the exposure of OHP-treated neurons to a neuroprotective stimulus, represented by a high dose of NGF, counteracts OHP-induced neuronal mortality. This effect was achieved by restoring the MAPK activation existing in untreated control cells. Increased viability occurred also after the administration of retinoic acid (RA), a pro-differentiative agent able to activate both JNK/Sapk and ERK1/2. The use of specific chemical inhibitors of MAPKs confirms the importance of this class of proteins for the neuroprotective pathway, since they reverse the protective effect. In summary, our findings assess the validity of MAPKs as the target of neuroprotective therapies during chemotherapeutic treatment. Moreover they also describe a double role for ERK1/2, depending on cellular stimulation, since it mediates neuronal apoptosis after OHP exposure. However, it is also important, as is JNK/Sapk, in preserving the correct cellular differentiation that is pivotal for neuronal survival.     

Elevation of intracellular calcium ions is essential for the H2O2-induced activation of SAPK/JNK but not for that of p38 and ERK in Chinese hamster V79 cells

The mitogen-activated protein kinases (MAPK), including stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and extracellular signal-related kinase (ERK), are believed to be important biomolecules in cell proliferation, survival, and apoptosis induced by extracellular stimuli. In Chinese hamster V79 cells exposed to hydrogen peroxide (H2O2), we recently demonstrated that SAPK/JNK was activated by tyrosine kinase and intracellular Ca2+ ([Ca2+]i). In this study, we report that [Ca2+]i release from intracellular stores is important in the activation of SAPK/JNK but not p38 and ERK. H2O2-induced elevation of [Ca2+]i was observed in Ca2+-free medium. Pretreatment with thapsigargin, a Ca2+-ATPase inhibition of endoplasmic reticulum (ER), did not influence H2O2-induced elevation of [Ca2+]i in the absence of external Ca2+. An intracellular Ca2+ chelator (BAPTA-AM) inhibited H2O2-induced phosphorylation of SAPK/JNK, but an extracellular Ca2+ chelator (EDTA) or a Ca2+ entry blocker (NiCl2) did not. Activation of p38 and ERK in V79 cells exposed to H2O2 was observed in the presence of these inhibitors. These results suggest that [Ca2+]i release from intracellular stores such as mitochondria or nuclei but not ER, occurred after H2O2 treatment and Ca2+-dependent tyrosine kinase-induced activation of SAPK/JNK, although [Ca2+]i was unnecessary for the H2O2-induced activation of p38 and ERK.     

C-Jun NH2 terminal kinase (JNK) is an essential mediator of Toll-like receptor 2-induced corneal inflammation

TLRs play an important role in the host inflammatory response to bacteria and bacterial products by activating a cascade of intracellular events leading to production of proinflammatory and chemotactic cytokines. To determine the role of MAPKs in TLR- induced corneal inflammation, we stimulated human corneal epithelial (HCE) cells with TLR2 ligands, tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3Cys) or inactivated Staphylococcus aureus, and examined the time course of expression of MAPKs and the effect of MAPK inhibition on IkBalpha degradation and CXC chemokine production. We found that S. aureus and Pam3Cys stimulate phosphorylation of JNK, p38 MAPK, and ERK within 4 h and that blockade of JNK, but not p38 or ERK phosphorylation, had an inhibitory effect on IkBalpha degradation and CXC chemokine production. To determine if JNK is also important in TLR2-induced corneal inflammation in vivo, we examined JNK1(-/-) mice and pharmacological inhibitors in a murine model of TLR2-induced corneal inflammation which is characterized by neutrophil recruitment to the corneal stroma and development of corneal haze. We found that corneal inflammation was significantly impaired in JNK1(-/-) mice compared with control mice, and in mice treated with the JNK inhibitor compared with vehicle control. Taken together with results from HCE cells, these findings demonstrate that JNK has an essential role in TLR2-induced corneal inflammation.     

Syk is required for p38 activation and G2/M arrest in B cells exposed to oxidative stress

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. In this study, we have investigated the role of Syk in p38 activation and the regulation of cell-cycle progression upon oxidative stress. In B cells, p38 is activated by hydrogen peroxide (H(2)O(2)) stimulation. Syk is required for p38 activation following stimulation with 10-100 microM H(2)O(2), but not with 1 mM H(2)O(2). H(2)O(2)-induced p38 activation is abrogated in phospholipase C-gamma2 (PLC-gamma2)-deficient as well as Syk-deficient cells, suggesting that Syk activates p38 through PLC-gamma2 upon H(2)O(2) stimulation. Although stimulation with 20-100 microM H(2)O(2) induces cellular apoptosis in B cells, pretreatment with SB203580, a p38-specific inhibitor, has no effect on H(2)O(2)-induced apoptosis. Flow cytometric analysis reveals that B cells exposed to 10-20 microM H(2)O(2) exhibit cell-cycle profile of G2/M arrest, and pretreatment with SB203580 inhibits only a little H(2)O(2)-induced G2/M arrest. On the other hand, Syk-deficient cells show no induction of G2/M arrest following H(2)O(2) stimulation. These findings indicate that Syk plays a role in the regulation of cell-cycle progression in G2/M phase via p38-dependent and -independent pathways after oxidative stress.     

Activation of c-Jun NH(2)-terminal kinase is required for porcine reproductive and respiratory syndrome virus-induced apoptosis but not for virus replication

Apoptosis of host cells plays a critical role in pathogenesis of virus infection. MAPK kinases especially stress-activated protein kinases c-Jun NH(2)-terminal kinase (SAPK/JNK) and p38 are often involved in virus-mediated apoptosis. It has been shown that porcine reproductive and respiratory syndrome virus (PRRSV) infection resulted in apoptosis of the host cells both in vitro and in vivo. The current investigation was initiated to determine whether stress-activated protein kinases JNK and p38 play a role in apoptosis induction by PRRSV infection. We examined phosphorylation of JNK and p38, and found that JNK but not p38 was activated in response to PRRSV infection. We then examined effects of this kinase on apoptosis induction and virus replication by using specific inhibitor. We found that JNK inhibition by its inhibitor SP600125 led to the abolishment of PRRSV-mediated apoptosis, but did not suppress virus replication. Further studies demonstrated that ROS generation was involved in JNK activation, and Bcl-2 family anti-apoptotic proteins Mcl-1 and Bcl-xl were downstream targets of JNK to mediate apoptosis. We conclude that activation of JNK signaling pathway is essential for PRRSV-mediated apoptosis but not for virus replication.     

KR-31378 protects cardiac H9c2 cells from chemical hypoxia-induced cell death via inhibition of JNK/p38 MAPK activation

Using a metabolic inhibition buffer as an ischemic model, we show here that KR-31378, a cardioselective ATP-sensitive potassium channel opener, protects H9c2 cells from chemical hypoxia (CH)-induced cell death. Our previous study showed that CH downregulated caspase activities, but led to differential activation of mitogen-activated protein kinases (MAPKs) in H9c2 cells. The repression of CH-induced c-jun N-terminal kinase (JNK)/p38 MAPK activation resulted in partial protection against CH- induced cell death, implying JNK/p38 MAPK's causative role in CH-induced cell death. This study furthers that research and examines if KR-31378's protective effect came from modulating MAPK activity and/or caspase activity in H9c2 cells. Although KR-31378 did not restore downregulated caspase-3 activity, it did block the activation of JNK and p38 MAPK in a dose-dependent manner. Extracellular signal-regulated kinase activity was not recovered by KR-31378 treatment. CH-induced reactive oxygen species (ROS) generation was suppressed by KR-31378. Thus our results indicate that the cardioprotective effect of KR-31378 in CH is due, at least in part, to the differential inhibition of MAPKs.     

Simultaneous induction of apoptotic and survival signaling pathways in macrophage-like THP-1 cells by Shiga toxin 1

Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-alpha) and produced soluble TNF-alpha after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-alpha, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (DeltaPsi(m)), and released cytochrome c into the cytoplasm. The DeltaPsi(m) values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-kappaB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.     

Effect of inhibition of extracellular signal-regulated kinase 1 and 2 pathway on apoptosis and bcl-2 expression in Helicobacter pylori-infected AGS cells

Helicobacter pylori induces activation of mitogen-activated protein kinases (MAPKs). However, its effect on H. pylori-induced apoptosis has not been evaluated. Thus, we examined whether H. pylori-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 MAPK activation affects gastric epithelial cell apoptosis and bcl-2 family gene expression, especially in relation to the cagA status of an H. pylori strain. In flow cytometric and oligonucleosome-bound DNA enzyme-linked immunosorbent assay analyses, infection with cagA(+) H. pylori strains induced gastric cancer cell apoptosis in AGS cells more prominently than infection with cagA mutants. Activation of ERK1/2 and p38 MAPKs was also more prominent in cagA(+) strains. Pretreatment with a MEK inhibitor (PD98059) inhibited ERK1/2 activation and increased H. pylori-induced apoptosis significantly. This increased apoptosis was accompanied by decreased antiapoptotic bcl-2 mRNA expression among bcl-2-related genes (bcl-2, bax, bak, mcl-1, and bcl-X(L/S)), and the effect was also more prominent in the cagA(+) strains. However, the alteration of bcl-2 gene expression was not accompanied by protein level changes. Inhibition of p38 using specific inhibitor SB203580 decreased H. pylori-induced apoptosis but resulted in little alteration of bcl-2-related gene expression. In conclusion, H. pylori-induced ERK1/2 activation, especially by the cagA(+) H. pylori strain, may play a protective role against gastric epithelial cell apoptosis partially through maintenance of bcl-2 gene expression.     

Specific inhibitors of mitogen-activated protein kinase and PI3-K pathways impair immune responses by hemocytes of trematode intermediate host snails

To characterize molecular mechanisms regulating snail cellular immune responses, the contributions of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3-K) were examined in hemocytes of the trematode intermediate host snails Biomphalaria glabrata and Lymnaea stagnalis. Simultaneous measurement of phagocytosis/encapsulation and H2O2 production by hemocytes in the presence or absence of specific signal transduction inhibitors was used to assess the role of extracellular-signal regulated kinases 1 and 2 (ERK1/2), p38, JNK and PI3-K. Hemocyte spreading was significantly reduced in a dose-dependent manner by the ERK inhibitor, PD098059, and by wortmannin, a potent PI3-K inhibitor. The JNK inhibitor, SP600125, and the p38 kinase inhibitor, SB203580, had no effect on hemocyte spreading. Sheep red blood cell phagocytosis was significantly impaired by PD098059, SP600125, and SB203580. Hydrogen peroxide production during phagocytosis was severely inhibited by PD098059. Additionally, PD098059, but not the other inhibitors, significantly impaired the cellular encapsulation of trematode larvae and H2O2 production during encapsulation. These results suggest that MAPK and PI3-K signal transduction pathways play a pivotal role in the immune responses of snail hemocytes. PI3-K and ERK appear to strongly regulate cell motility. ERK, JNK and p38 contribute to phagocytosis-mediated signal transduction. ERK also play a major role in oxidative burst activation and the encapsulation of trematode larvae by snail hemocytes.     

Apoptosis triggered by phagocytosis-related oxidative stress through FLIPS down-regulation and JNK activation

Tumor necrosis factor-alpha (TNF-alpha)-activated neutrophils phagocytose and eliminate bacteria by using such oxidants as hydrogen peroxide (H(2)O(2)) and hypochlorous acid (HOCl), which is produced from H(2)O(2) by myeloperoxidase (MPO). Thereafter, neutrophils eventually undergo apoptosis to prevent excessive inflammation. However, it is unclear how this process is regulated. Here, we show that cotreatment of TNF-alpha-resistant neutrophilic HL-60 cells with taurine chloramine (TauCl), a detoxified form of HOCl, and TNF-alpha renders them susceptible to apoptosis, mostly by preventing nuclear factor-kappaB (NF-kappaB) activation. Of several NF-kappaB target genes tested, FLICE inhibitory protein short form (FLIP(S)) was specifically down-regulated by TauCl. TNF-alpha/TauCl cotreatment-induced apoptosis was largely blocked by stable expression of FLIP(S). Cotreatment with TNF-alpha and H(2)O(2) promoted apoptotic signaling via MPO activation and subsequent attenuation of FLIP(S) expression. TNF-alpha priming with H(2)O(2) or bacteria caused MPO-dependent apoptosis in human neutrophils. However, FLIP(S) knock-down by siRNA did not affect the viability of cells treated with TNF-alpha, implying that TauCl may affect another pathway in TNF-alpha-driven apoptosis. Indeed, oxidization of thioredoxin-1 (Trx-1) by TauCl induced the activation of apoptosis signal-regulating kinase 1 (ASK1) and cJun N-terminal kinase (JNK), thereby triggering TNF-alpha-mediated apoptosis. Taken together, these results indicate that the antiapoptotic signaling induced by TNF-alpha via NF-kappaB activation can be altered to promote apoptosis via H(2)O(2)-MPO-mediated FLIP(S) down-regulation and JNK activation.     

Inflammatory response under zinc deficiency is exacerbated by dysfunction of the T helper type 2 lymphocyte–M2 macrophage pathway

Nutritional zinc deficiency leads to immune dysfunction and aggravates inflammation. However, the underlying mechanism remains unknown. In this study, the relationship between macrophage subtypes (M1 and M2) and helper T lymphocytes (Th1 and Th2) was investigated using the spleen from rats fed zinc-deficient or standard diet. In experiment I, 5-week-old male Sprague-Dawley rats were fed a zinc-deficient diet (without zinc additives) or a standard diet (containing 0·01% zinc) for 6 weeks. In experiment II, the rats were divided into four groups: one group was fed a standard diet for 6 weeks; two groups were fed zinc-deficient diets and were injected three times a week with either saline or interleukin-4 (IL-4) (zinc-deficient/IL-4 i.p.); a fourth group (zinc-deficient/standard) was fed a zinc-deficient diet for 6 weeks followed by a standard diet for 4 weeks. In experiment I; GATA-binding protein 3 (GATA-3) protein level, M2 macrophage, CD3⁺ CD8⁺ cells, and IL-4/IL-13-positive cells significantly decreased in the spleens of the zinc-deficient group. Additionally, IL-1β and macrophage inflammatory protein-1α (MIP-1α) mRNA levels significantly increased in the splenic macrophages of the zinc-deficient group. In experiment II; M2 macrophages, CD3⁺ CD8⁺ cells, IL-4/IL-13-positive cells, and GATA-3 protein levels significantly increased in the spleens of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Furthermore, IL-1β and MIP-1α mRNA levels decreased in the splenic macrophages of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Zinc deficiency-induced aggravated inflammation is related to Th2 lymphocytes and followed by the association with loss of GATA-3, IL-4 and anti-inflammatory M2 macrophages. Importantly, IL-4 injection or zinc supplementation can reverse the effects of zinc deficiency on immune function.