Tyrosine kinase but not phospholipid/Ca2+ signaling pathway is involved in interferon-γ stimulation of I-a expression in macrophages

The specific signal transduction pathway(s) involved in the induction of the expression of the MHC class II molecule, la, on macrophages by interferon-γ (IFN-γ) is unclear. In this paper, we assessed the role of several signal transduction pathways including calcium mobilization, phospholipase C, protein kinase C and cyclic nucleotide-dependent protein kinase, and the tyrosine kinase pathways. IFN-γ was unable to mobilize intracellular calcium, unlike platelet-activating factor, which stimulated a threefold increase in cytosolic Ca²⁺ concentration in macrophages. Inhibition of the phospholipase C pathway by U73122 or ET-180CH₃ and of phosphatidic acid phosphohydrolase by propranolol did not suppress IFN-γ-induced la expression. In addition, inhibition of protein kinase C by calphostin C or cyclic nucleotide-dependent protein kinase by HA1004 did not suppress la expression. However, IFN-γ-induced la expression was significantly suppressed when the tyrosine kinase pathway was inhibited with herbimycin A and genestein. In addition, those two inhibitors suppressed tyrosine phosphorylation of several proteins in macrophages that may or may not be involved in the induction of la expression. Thus, IFN-γ used only the tyrosine kinase signaling pathway, but not the phospholipid/Ca²⁺ signaling pathways, to induce la expression in macrophages. © 1996 Wiley-Liss, Inc.     

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.     

Paralytic peptide activates insect humoral immune response via epidermal growth factor receptor

Paralytic peptide (PP) activates innate immunity of silkworm Bombyx mori, inducing production of anti-microbial peptides (AMPs) and phagocytosis-related proteins; however the signal pathways of PP-dependent immune responses are not clear. In present study, we characterized BmE cells as a PP-responsive cell line by examining the expression of AMP genes and activation of p38 mitogen-activated protein kinase (p38 MAPK) under PP stimulation, and we also found PP directly binds to BmE cell membrane. Then we found that PP-dependent expression of AMP genes is suppressed by tyrosine kinase inhibitor (genistein) both in BmE cells and in fat body of silkworm larvae. Moreover, the specific tyrosine kinase epidermal growth factor receptor (EGFR) inhibitor (AG1478) attenuates PP-induced expression of AMP genes in BmE cells and fat body of silkworm and RNA interference (RNAi) to BmEGFR also suppresses PP-induced expression of AMP genes. Furthermore, the PP-induced p38 MAPK phosphorylation is inhibited by AG1478. Our results suggest that BmE cells can be used as a cell model to investigate the signal pathway of PP-dependent humoral immune response and receptor tyrosine kinase EGFR/p38 MAPK pathway is involved in the production of AMPs induced by PP.     

Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A₂ via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca²⁺]_i) in MHS macrophage cells. Ionomycin increased [Ca²⁺]_i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca²⁺→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca²⁺ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca²⁺→ ERK pathway, which plays a key role in suppression of inflammatory reactions.     

Cytochemical Alterations in the Rat Retina by LPS Administration

LPS-induced inflammation and changes in protein phosphorylation and the JAK-STAT pathway accompanying glial activation after LPS treatment, were followed by analyzing secreted proinflammatory cytokine levels. The administration of LPS caused tyrosine phosphorylation of STAT3 in retinae and induced glial fibrillary acidic protein. (GFAP) from the nerve fiber layer to the ganglion cell layer. Our results suggest that the LPS-induced activation of the JAK2/STAT3 signaling pathway may play a key role in the induction of astrogliosis. However, no significant increase in vimentin, OX-42 or inducible nitric oxide synthase (iNOS) expressions were observed after LPS administration. Sphingosine kinase catalyzes the conversion of sphingosine to sphingosine-1–phosphate (So-1-P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, it was found that sphingolipid metabolite levels were elevated in the serum and retinae of LPS-injected rats. To further investigate the chronic effect of increased So-1-P in the retina, So-1-P was infused intracerebroventricularly (i.c.v.) into rats using an osmotic minipump at 100 pmol/10 μl h^-1 for 7 days, and was found to increase retinal GFAP expression. These observations suggest that LPS induces the activation of retinal astrocytes via JAK2/STAT3 and that LPS affects So-1-P generation. Our findings also suggest that elevated So-1-P in the retina and/or in serum could induce cytochemical alterations in LPS treated or inflamed retinae.     

Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages.

Bacterial lipopolysaccharide (LPS) is a potent activator of antibacterial responses by macrophages. Following LPS stimulation, the tyrosine phosphorylation of several proteins is rapidly increased in macrophages, and this event appears to mediate some responses to LPS. We now report that two of these tyrosine phosphoproteins of 41 and 44 kDa are isoforms of mitogen-activated protein (MAP) kinase. Each of these proteins was reactive with anti-MAP kinase antibodies and comigrated with MAP kinase activity in fractions eluted from a MonoQ anion-exchange column. Following LPS stimulation, column fractions containing the tyrosine phosphorylated forms of p41 and p44 exhibited increased MAP kinase activity. Inhibition of LPS-induced tyrosine phosphorylation of these proteins was accompanied by inhibition of MAP kinase activity. Additionally, induction of p41/p44 tyrosine phosphorylation and MAP kinase activity by LPS appeared to be independent of activation of protein kinase C, even though phorbol esters also induced these responses. These results demonstrate that LPS induces the tyrosine phosphorylation and activation of at least two MAP kinase isozymes. Since MAP kinases appear to modulate cellular processes in response to extracellular signals, these kinases may be important targets for LPS action in macrophages.     

Inhibitory Effects of KP-A159, a Thiazolopyridine Derivative,on Osteoclast Differentiation,Function, and Inflammatory Bone Loss via Suppression of RANKL-Induced MAP Kinase Signaling Pathway

Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs), but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase1/2 (MEK1/2). In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS)-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss.     

Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor.

Signaling through the T cell antigen receptor (TCR) results both in rapid increases in tyrosine phosphorylation on a number of proteins and in the activation of the phosphatidylinositol pathway. It is not clear how stimulation of the TCR leads to these signaling events. Mutants of the Jurkat T cell line have been previously isolated that fail to show increases in calcium following receptor stimulation. Analysis of one of these mutants, JCaM1, which is defective in the induction of tyrosine phosphorylation, revealed a defect in the expression of functional lck tyrosine kinase. The lack of lck activity was caused in part by a splicing defect. Expression of the lck cDNA in JCaM1 restores the ability of the cell to respond to TCR stimulation. These results indicate that lck is required for normal signal transduction through the TCR.     

Receptor-mediated activation of human B lymphocytes in a nonphosphotyrosine-dependent manner.

The B cell AgR regulates two signal transduction pathways: the tyrosine kinase and the phosphatidylinositol (PtdIns) pathways. Stimulation of B cells with Ag or anti-Ig antibody results in a rapid increase in tyrosine phosphorylation of multiple substrates. The AgR also mediates the activation of phospholipase C-gamma 1 (PLC-gamma 1) thus producing the second messengers, inositol trisphosphate and diacylglycerol. Although the detailed relationship between these two signaling pathways remains unclear, it has recently become apparent that PLC-gamma 1 might be a target for the AgR-associated protein tyrosine kinase. To address the question of whether tyrosine kinase activity is essential for B cell activation, we studied early biochemical changes and later cellular events induced by ligation of the purinoceptor (P2R). Ligation of ATP to its receptor on B cells has been previously shown to elicit increases in cytosolic free Ca2+ and inositol phosphate production as well as induction of c-fos mRNA expression and increased expression of IL-2 and transferrin receptors. We show here that ATP in a wide range of concentrations did not increase protein tyrosine kinase activity. In contrast with the AgR, P2R did not mediate tyrosine phosphorylation of PLC-gamma 1, thus suggesting that it may use another phosphoinositide-specific PLC that does not require phosphorylation on tyrosine residues for its activation. The results were supported by experiments with a specific tyrosine kinase inhibitor, tyrphostin AG-126. Preincubation with this inhibitor blocked AgR but not P2R-mediated inositol phosphate production, cytosolic free Ca2+ changes, and IL-2 and transferrin receptor expression. The results indicate that the PtdIns pathway may be sufficient to induce activation of B cells and that the tyrosine phosphorylation pathway is not necessary for nonantigenic B cell activation.     

Regulation of homocysteine-induced MMP-9 by ERK1/2 pathway

Homocysteine (Hcy) induces matrix metalloproteinase (MMP)-9 in microvascular endothelial cells (MVECs). We hypothesized that the ERK1/2 signaling pathway is involved in Hcy-mediated MMP-9 expression. In cultured MVECs, Hcy induced activation of ERK, which was blocked by PD-98059 and U0126 (MEK inhibitors). Pretreatment with BAPTA-AM, staurosporine (PKC inhibitor), or Gö6976 (specific inhibitor for Ca²⁺-dependent PKC) abrogated ERK phosphorylation, suggesting the role of Ca²⁺ and Ca²⁺-dependent PKC in Hcy-induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G protein-coupled receptors (GPCRs) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVECs with genistein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting the involvement of the PTK pathway in Hcy-induced ERK activation, which was mediated by intracellular Ca²⁺ pool depletion. ERK activation was attenuated by preincubation with N-acetylcysteine (NAC) and SOD, suggesting the role of oxidation in Hcy-induced ERK activation. Pretreatment with an ERK1/2 blocker (PD-98059), staurosporine, folate, or NAC modulated Hcy-induced MMP-9 activation as measured using zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVECs. calcium signaling; protein kinase C; Src; G protein-coupled receptor; nonreceptor tyrosine kinase; protein G_i; protein G_q; protein tyrosine kinase 2; microvascular endothelial cell; cardiovascular remodeling     

Lipopolysaccharide-Induced NF-κB Activation in Human Endothelial Cells Involves Degradation of IκBα but Not IκBβ

We studied the signal transduction pathways involved in NF-κB activation and the induction of the cytoprotective A20 gene by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). LPS induced human A20 mRNA expression with a maximum level 2 h after stimulation. The proteasome inhibitorN-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocked A20 mRNA expression and partially inhibited NF-κB DNA-binding activity induced by LPS treatment. LPS induced IκBα degradation at 30–60 min after treatment, but did not induce IκBβ degradation up to 120 min. In contrast, TNF-α rapidly induced IκBα degradation within 5 min and IκBβ degradation within 15 min. Cycloheximide did not prevent LPS-induced IκBα degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated IκBα degradation. LPS-induced IκBα degradation was inhibited by ALLN, confirming that ALLN inhibits NF-κB activation by preventing IκBα degradation. Of note, HMA also inhibited LPS-induced IκBα degradation. However, tyrosine phosphorylation of IκBα itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of IκBα is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-κB-dependent genes through degradation of IκBα, not IκBβ, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of IκBα.     

Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-kappa B activation in human peripheral blood monocytes

Bacterial lipopolysaccharide (LPS) is a powerful activator of the innate immune system. Exposure to LPS induces an inflammatory reaction in the lung mediated primarily by human blood monocytes and alveolar macrophages, which release an array of inflammatory chemokines and cytokines including IL-8, TNF-alpha, IL-1beta, and IL-6. The signaling mechanisms utilized by LPS to stimulate the release of cytokines and chemokines are still incompletely understood. Pretreatment with the protein tyrosine kinase-specific inhibitors genistein and herbimycin A effectively blocked LPS-induced NF-kappaB activation as well as IL-8 gene expression in human peripheral blood monocytes. However, when genistein was added 2 min after the addition of LPS, no inhibition was observed. Utilizing a coimmunoprecipitation assay, we further showed that LPS-stimulated tyrosine phosphorylation of Toll-like receptor 4 (TLR4) may be involved in downstream signaling events induced by LPS. These findings provide evidence that LPS-induced NF-kappaB activation and IL-8 gene expression use a signaling pathway requiring protein tyrosine kinase and that such regulation may occur through tyrosine phosphorylation of TLR4.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

The Novel Secreted Adipokine WNT1-inducible Signaling Pathway Protein 2 (WISP2) Is a Mesenchymal Cell Activator of Canonical WNT

WNT1-inducible-signaling pathway protein 2 (WISP2) is primarily expressed in mesenchymal stem cells, fibroblasts, and adipogenic precursor cells. It is both a secreted and cytosolic protein, the latter regulating precursor cell adipogenic commitment and PPARγ induction by BMP4. To examine the effect of the secreted protein, we expressed a full-length and a truncated, non-secreted WISP2 in NIH3T3 fibroblasts. Secreted, but not truncated WISP2 activated the canonical WNT pathway with increased β-catenin levels, its nuclear targeting phosphorylation, and LRP5/6 phosphorylation. It also inhibited Pparg activation and the effect of secreted WISP2 was reversed by the WNT antagonist DICKKOPF-1. Differentiated 3T3-L1 adipose cells were also target cells where extracellular WISP2 activated the canonical WNT pathway, inhibited Pparg and associated adipose genes and, similar to WNT3a, promoted partial dedifferentiation of the cells and the induction of a myofibroblast phenotype with activation of markers of fibrosis. Thus, WISP2 exerts dual actions in mesenchymal precursor cells; secreted WISP2 activates canonical WNT and maintains the cells in an undifferentiated state, whereas cytosolic WISP2 regulates adipogenic commitment.     

Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a G alpha i3/Ras/Raf/MAPK pathway

We have previously reported that angiotensin II (ANG II) stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which, in turn, activates MAPK, resulting in an increase in nitric oxide synthase (NOS) expression in pulmonary artery endothelial cells (PAECs). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and an increase in NOS protein in PAECs. Transfection of PAECs with G_i3 dominant negative (DN) cDNA blocked the ANG II-dependent activation of Src, ERK1/ERK2 phosphorylation, and increase in NOS expression. ANG II stimulated an increase in tyrosine phosphorylation of sequence homology of collagen (Shc; 15 min) that was prevented when PAECs were pretreated with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-D]pyrimidine (PP2), a Src inhibitor. ANG II induced a Src-dependent association between Shc and growth factor receptor-bound protein 2 (Grb2) and between Grb2 and son of sevenless (Sos), both of which were maximal at 15 min. The ANG II-dependent increase in Ras GTP binding was prevented when PAECs were pretreated with the AT₂ antagonist PD-123319 or with PP2 or were transfected with Src DN cDNA. ANG II-dependent activation of MAPK and the increase in endothelial NOS (eNOS) were prevented when PAECs were transfected with Ras DN cDNA or treated with FTI-277, a farnesyl transferase inhibitor. ANG II induction of Raf-1 phosphorylation was prevented when PAECs were pretreated with PD-123319 and PP2. Raf kinase inhibitor 1 prevented the ANG II-dependent increase in eNOS expression. Collectively, these data suggest that G_i3, Shc, Grb2, Ras, and Raf-1 link Src to activation of MAPK and to the AT₂-dependent increase in eNOS expression in PAECs. Src; mitogen-activated protein kinase     

Plasminogen activator inhibitor type 1 expression induced by lipopolysaccharide of Porphyromonas gingivalis in human gingival fibroblast

In the gingival tissues of patients with periodontitis, inflammatory responses are mediated by a wide variety of genes. In our previous screening study, plasminogen activator inhibitor type 1 (PAI-1) mRNA binding protein expression was increased in gingiva from periodontitis patients. In this study, we further investigated the signaling pathway involved in PAI-1 expression induced by Porphyromonas gingivalis LPS (Pg LPS) in human gingival fibroblasts (HGF). When HGFs were treated with Pg LPS, both PAI-1 mRNA expression and PAI-1 protein were induced in a dose-dependent manner. Pg LPS induced NF-κB activation and the expressions of PAI-1 mRNA and protein were suppressed by pretreating with a NF-κB inhibitor. Pg LPS also induced ERK, p38, and JNK activation, and Pg LPS-induced PAI-1 expression was inhibited by ERK/p38/JNK inhibitor pretreatment. In conclusion, Pg LPS induced PAI-1 expression through NF-κB and MAP kinases activation in HGF.     

Formyl-Met-Leu-Phe induces calcium-dependent tyrosine phosphorylation of Rel-1 in neutrophils

Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca²⁺ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105 kDa protein (Rel-1) requires Ca²⁺-dependent tyrosine phosphorylation. A rapid and time-dependent tyrosine phosphorylation of Rel-1 occurred following formyl-Met-Leu-Phe (fMLP) stimulation of human PMNs at concentrations that primed or activated the NADPH oxidase (10⁻⁹ to 10⁻⁶ M), becoming maximal after 30 s. Pretreatment with pertussis toxin (Ptx) or tyrosine kinase inhibitors abrogated this phosphorylation and inhibited fMLP activation of the oxidase. The fMLP concentrations employed also caused a rapid increase in cytosolic Ca²⁺ but chelation negated the effects, including the cytosolic Ca²⁺ flux, oxidase activation, and the tyrosine phosphorylation of Rel-1. Conversely, chelation of extracellular Ca²⁺ decreased the fMLP-mediated Ca²⁺ flux, had no affect on the oxidase, and augmented tyrosine phosphorylation of Rel-1. Phosphorylation of Rel-1 was inhibited when PMNs were preincubated with a p38 MAP kinase (MAPK) inhibitor (SB203580). In addition, fMLP elicited rapid activation of p38 MAPK which was abrogated by chelation of cytosolic Ca²⁺. Thus, fMLP concentrations that prime or activate the oxidase cause a rapid Ca²⁺-dependent tyrosine phosphorylation of Rel-1 involving p38 MAPK activation.