Fyn kinase mediates cortical actin ring depolymerization required for mast cell migration in response to TGF‐β in mice

Transforming growth factor‐β (TGF‐β) is a potent mast cell (MC) chemoattractant able to modulate local inflammatory reactions. The molecular mechanism leading to TGF‐β‐directed MC migration is not fully described. Here we analyzed the role of the Src family protein kinase Fyn on the main TGF‐β‐induced cytoskeletal changes leading to MC migration. Utilizing bone marrow‐derived mast cells (BMMCs) from WT and Fyn‐deficient mice we found that BMMC migration to TGF‐β was impaired in the absence of the kinase. TGF‐β caused depolymerization of the cortical actin ring and changes on the phosphorylation of cofilin, LIMK and CAMKII only in WT cells. Defective cofilin activation and phosphorylation of regulatory proteins was detected in Fyn‐deficient BMMCs and this finding correlated with a lower activity of the catalytic subunit of the phosphatase PP2A. Diminished TGF‐β‐induced chemotaxis of Fyn‐deficient cells was also observed in an in vivo model of MC migration (bleomycin‐induced scleroderma). Our results show that Fyn kinase is an important positive effector of TGF‐β‐induced chemotaxis through the control of PP2A activity and this is relevant to pathological processes that are related to TGF‐β‐dependent mast cell migration.     

Lyn deficiency affects B-cell maturation as well as survival

Lyn, an Src-family protein tyrosine kinase expressed in B lymphocytes, contributes to initiation of BCR signaling and is also responsible for feedback inhibition of BCR signaling. Lyn-deficient mice have a decreased number of follicular B cells and also spontaneously develop a lupus-like autoimmunity. We used flow cytometric analysis, BrdU labeling and our mathematical models of B-cell population dynamics, to analyze how Lyn deficiency impacts B-cell maturation and survival. We found that Lyn-deficient transitional 1 (T1) cells develop normally, but T2 cells develop primarily from the T1 subset in the spleen and fail to also develop directly from BM immature B cells. Lyn-deficient T2 cells either mature to the follicular B-cell type at a close to normal rate, or die in this compartment rather than access the T3 anergic subset. The ≈ 40% of WT follicular cells that were short-lived exited primarily by joining the T3 anergic subset, whereas the ≈ 15% Lyn(-/-) follicular cells that were not long lived had a high death rate and died in this compartment rather than entering the T3 subset. We hypothesize that exaggerated BCR signaling resulting from weak interactions with self-antigens is largely responsible for these alterations in Lyn-deficient B cells.     

Intracellular signaling pathways in IgE-dependent mast cell activation

Mast cells (MCs) are both central effectors and signaling cells in allergic reactions. Their key role in the immunopathology of asthma and other allergic diseases has been well documented. Molecular events leading to MC activation have not been yet fully established, however. Recent studies emphasize the key role of the protein tyrosine kinases Lyn and Fyn in MC signal transduction. The finding that Lyn kinase negatively regulates MC degranulation and that Fyn kinase enhances this effector response is of great importance. This creates new possibilities for therapeutic intervention in asthma and other allergic diseases. This review summarizes current knowledge on MC intracellular signaling and discusses the most recent strategies for the treatment of allergic diseases based on MC signaling pathway inhibition.     

Phospholipase C-β3 regulates FcɛRI-mediated mast cell activation by recruiting the protein phosphatase SHP-1

Mast cells are major effectors in high-affinity IgE receptor (Fc?RI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-β3 is crucial for Fc?RI-mediated mast cell activation. Plcb3(-/-) mice showed blunted Fc?RI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, Fc?RI stimulation of Plcb3(-/-) mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3(-/-) cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-β3 constitutively interacts with Fc?RI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-β3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, Fc?RI-mediated phenotypes were similar in Plcb3(-/-) and SHP-1 mutant mast cells. Thus, we have defined a PLC-β3- and SHP-1-mediated signaling pathway for Fc?RI-mediated cytokine production.     

Gain- and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in the myeloid lineage.

To investigate the role of the Lyn kinase in establishing signaling thresholds in hematopoietic cells, a gain-of-function mutation analogous to the Src Y527F-activating mutation was introduced into the Lyn gene. Intriguingly, although Lyn is widely expressed within the hematopoietic system, these mice displayed no propensity toward hematological malignancy. By contrast, analysis of aging cohorts of both loss- and gain-of-function Lyn mutant mice revealed that Lyn(-/-) mice develop splenomegaly, increased numbers of myeloid progenitors, and monocyte/macrophage (M phi) tumors. Biochemical analysis of cells from these mutants revealed that Lyn is essential in establishing ITIM-dependent inhibitory signaling and for activation of specific protein tyrosine phosphatases within myeloid cells. Loss of such inhibitory signaling may predispose mice lacking this putative protooncogene to tumorigenesis.     

Rottlerin-independent attenuation of pervanadate-induced tyrosine phosphorylation events by protein kinase C-delta in hemopoietic cells

The understanding and control of many pathophysiological conditions is based on knowledge of subtly regulated intracellular signaling networks. We have found that in pervanadate (PV)-treated J558L myeloma cells, amongst other signaling proteins, protein kinase C (PKC)-delta and src homology 2-containing inositol phosphatase (SHIP) are tyrosine phosphorylated on expression of the B cell receptor, suggesting a role for these proteins in the preformed B cell receptor transducer complex. Rottlerin, a widely used PKC-delta-specific inhibitor, efficiently blocks these PV-induced tyrosine phosphorylation events. Furthermore, PV treatment of bone marrow-derived mast cells (BMMC) also results in tyrosine phosphorylation of PKC-delta, SHIP, and additional proteins. Rottlerin also inhibits these responses, indicating that PKC-delta might play an important enhancing role in the propagation of phosphotyrosine signals in B cells and mast cells and hence in the regulation of function of both cell types. Therefore, BMMC from PKC-delta -/- mice were generated by in vitro differentiation and assayed for tyrosine phosphorylation events in response to PV. Intriguingly, and opposite to the Rottlerin data, PKC-delta -/- BMMC show a stronger response to PV than wild-type cells, suggesting an attenuating role for PKC-delta. This response can be inhibited equally well by Rottlerin, indicating clearly that Rottlerin is not specific for PKC-delta in vivo. A comparison between Rottlerin and the panspecific PKC inhibitor bisindolylmaleimide suggests that Rottlerin also targets kinases beyond the PKC family. Moreover, Ser473 phosphorylation of protein kinase B (PKB) after PV treatment is blocked by Rottlerin as efficiently as by the phosphatidylinositol 3-kinase inhibitor LY294002. In this report, we provide evidence that PKC-delta constitutes a crucial attenuating factor in B cell and mast cell signal transduction and suggest that PKC-delta is important for the regulation of physiological B and mast cell functions as well as for their pathophysiology. Furthermore, dominant PKC-delta-independent effects of Rottlerin are presented, indicating restrictions of this inhibitor for use in signal transduction research.     

Rat mucosal mast cells: the cultured bone marrow-derived mast cell is biochemically and functionally analogous to its counterpart in vivo.

Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow-derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor-alpha (TNF-alpha) storage and secretion, and TNF-alpha-dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two-dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC-associated enzyme, rat mast cell proteinase-2 (RMCP-2), but not RMCP-1, mast cell proteinase-5 (MCP-5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the periotoneal MC (PMC). BMMC demonstrated levels of TNF-alpha-dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF-alpha, in comparison with PMC, but both MC types generated substantial amounts of TNF-alpha 6 hr following IgE-mediated activation. Pretreatment of PMC with recombinant rat interferon-gamma (IFN-gamma) for 20 hr inhibited anti-immunoglobulin E (anti-IgE)-mediated release of the granule-associated enzyme, beta-hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC.     

Protein Tyrosine Phosphatase ε is a Negative Regulator of FcεRI-mediated Mast Cell Responses

Modulation of mast-cell activation may provide novel ways to control allergic diseases. Here, we show that protein tyrosine phosphatase ε (PTPε; Ptpre ) plays key regulatory roles during mast-cell activation mediated by the high-affinity IgE receptor (FcεRI). Bone marrow-derived mast cells (BMMC) from Ptpre ^−/− mice exhibited enhanced FcεRI-induced Ca²⁺ mobilization and mitogen-activated protein kinase (MAPK) (JNK and p38) activation, and showed corresponding enhancement of evoked degranulation and cytokine production, but not leukotriene production. Examination of proteins linking tyrosine kinase activation and Ca²⁺ mobilization revealed that the absence of PTPε leads to increased phosphorylation of the linker for activation of T cells and SH2 domain-containing leucocyte phosphoproteins of 76 kDa, but not Grb2-associated binder-2 (Gab2). Because Gab2 is considered to be situated downstream of Fyn kinase, we reasoned that Fyn may not be a target of PTPε. In the event, Syk but not Lyn was hyperphosphorylated in PTPε-deficient BMMC. Thus, PTPε most likely exerts its effects at the level of Syk, inhibiting downstream events including phosphorylation of SLP-76 and linker of activated T cells and mobilization of Ca²⁺. Consistent with the in vitro data, antigen- and IgE-mediated passive systemic anaphylactic reactions were augmented in Ptpre ^−/− mice. Given that the number of mast cells is unchanged in these mice, this observation most likely reflects alterations of mast cell-autonomous signalling events. These data suggest that PTPε negatively regulates FcεRI-mediated signalling pathways and thus constitutes a novel target for ameliorating allergic conditions.     

Positive and negative regulation of Fc epsilon receptor I-mediated signaling events by Lyn kinase C-terminal tyrosine phosphorylation

Although it is known that the Src family tyrosine kinase Lyn initiates Fc epsilon receptor I (Fc epsilon RI) signaling by phosphorylation of the receptor subunits, regulation of Lyn kinase activity and its consequences for receptor signaling are incompletely understood. Using a phospho-Lyn-specific antiserum, we show an increased phosphorylation of the Lyn C-terminal regulatory tyrosine and decreased Lyn kinase activity during Fc epsilon RI-mediated mast cell activation. Mutant Lyn, defective in the C-terminal tyrosine, constitutively phosphorylated several substrates in resting cells, but did not cause Fc epsilon RI internalization or spontaneous degranulation. Fc epsilon RI-induced signaling in the presence of constitutively active Lyn exhibited enhanced phosphorylation of the receptor subunits, Syk, LAT, Gab2, phospholipase C (PLC)gamma 1 and PLC gamma 2, and production of phosphatidylinositol 3,4,5-trisphosphate. Although enzymatic activities of PLC gamma 1 and PLC gamma 2 were also up-regulated, amounts of inositol 1,4,5-trisphosphate, mobilization of intracellular calcium and degranulation were suppressed. Additionally, constitutively active Lyn was strikingly less efficient than wild-type Lyn in restoring the receptor-mediated calcium responses in bone marrow mast cells derived from Lyn(-/-) mice. These findings pinpoint the tight regulation of Lyn kinase activity as a critical event in mast cell degranulation.     

The PH domain adaptor protein Bam32/DAPP1 functions in mast cells to restrain FcɛRI-induced calcium flux and granule release

Mast cell activation triggered by IgE binding to its high affinity receptor Fc?RI is highly dependent on signaling via phosphoinositde 3-kinases (PI3K). The phosphoinositide phosphatase SHIP controls mast cell activation by regulating accumulation of D3 phosphoinositide second messengers generated by PI3K. The PH domain adaptor protein Bam32/DAPP1 binds specifically to the D3 phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 (the substrate and product of SHIP respectively). In B cells, Bam32 is phosphorylated by Src family kinases including Lyn, and is required for antigen receptor-induced activation; however the function of Bam32 in mast cells is unknown. Here we report that Bam32 is expressed in mast cells, is recruited to the plasma membrane upon stimulation and functions in Fc?RI signaling. Examination of bone marrow-derived mast cells (BMMC) isolated from Bam32-deficient mice revealed enhanced Fc?RI-induced degranulation and IL-6 production, indicating that Bam32 may function to restrain signaling via Fc?RI. These enhanced degranulation responses were PI3K-dependent, as indicated by blockade with PI3K inhibitors wortmannin or IC87114. While Bam32-deficient BMMC showed reduced Fc?RI-induced activation of mitogen-activated protein kinases ERK and JNK, Fc?RI-induced calcium flux and phosphorylation of PLCγ1 and Akt were increased. Bam32-deficient BMMC showed significantly reduced phosphorylation of Lyn and SHIP, indicating reduced activity of inhibitory signaling pathways. Together our results identify Bam32 as a novel regulator of mast cell activation, potentially functioning in membrane-proximal integration of positive and negative signaling pathways.     

Microenvironmental regulation of inducible nitric oxide synthase expression and nitric oxide production in mouse bone marrow-derived mast cells

In addition to its well-known role in relaxation of vascular smooth muscle, NO modulates immune responses in a concentration- and location-specific manner. For MC, it is well accepted that exogenous NO regulates their function. However, there are inconsistencies in the literature of whether MC express NOS and make NO. MC progenitors mature in peripheral tissues, but the factors that influence MC maturation and their specific phenotype, such as whether they express NOS, are not well understood. To study microenvironmental conditions that could be "permissive" for NOS expression, we cultured BMMC in various conditions--BMMC(IL-3), BMMC(SCF/IL-3), or BMMC(SCF/IL-4)-for >3 weeks and examined NOS expression. We detected Nos2 mRNA in BMMC(SCF/IL-4) but not BMMC(IL-3) or BMMC(SCF/IL-3). After stimulation with IFN-γ and/or LPS, NOS2 expression and NO production were detected in BMMC(SCF/IL-4) but rarely detected in BMMC cultured with other conditions. Confocal microscopic analysis showed that NOS2 expression induced by IFN-γ colocalized in CD117(+) BMMC. NO production, after activation with IFN-γ and LPS in BMMC(SCF/IL-4), was abrogated by pretreatment with the NOS2-specific inhibitor. In addition to NOS2 expression, BMMC(SCF/IL-4) were distinguished from BMMC(IL-3) in heparin and MMCP expression. Thus, MC progenitors that develop in SCF + IL-4 can be induced to express NOS2 after receiving appropriate signals, such as IFN-γ, and subsequently produce NO. Microenvironmental conditions during their development can influence whether MC are capable of NOS expression and of NO production.     

Secretory lysosomes of mouse mast cells store and exocytose active caspase‐3 in a strictly granzyme B dependent manner

In this study, we report that cytoplasmic granules from in vivo and in vitro derived mouse mast cells (MCs) contain active granzyme B (gzmB) and caspase‐3, which is consistent with recent findings. Studying WT and gzmB‐deficient mice, we observed that BM‐derived MCs (BMMCs) from both strains contain cytosolic pro‐caspase‐3, but only WT BMMCs expressed active caspase‐3 limited to their secretory lysosomes. Confocal microscopy revealed colocalization of active caspase‐3 and gzmB in these cytoplasmic granules. The combined data demonstrate that the generation and storage of active caspase‐3 is gzmB‐dependent. The finding that BMMCs secrete caspase‐3 and gzmB after Ag stimulation suggests that both proteases contribute to extracellular MC‐mediated proteolytic events. Although the extracellular function of MC‐derived caspase‐3 remains unclear, we show that BMMC‐secreted caspase‐3 cleaves IL‐33, a cytokine that contributes to the development of asthma and arthritis. We also show that an in vitro propagated cytolytic T‐lymphocyte line constitutively expresses gzmB together with active caspase‐3, suggesting a novel interaction of these proteases in the execution of multiple innate and adaptive immune responses.     

The tyrosine kinase Lyn is required for B cell development beyond the T1 stage in the spleen: rescue by over-expression of Bcl-2

We have analyzed the effects of deficiency in the tyrosine kinase Lyn on B cell development using transgenic mice that express a B cell antigen receptor (BCR) of defined specificity (3-83,anti-H-2K(k or b)). In the absence of Lyn, immature B cells are abundant in the bone marrow and spleen up until the T1 stage (IgM(hi) IgD(-) CD21(-)CD23(-)), after which B cell development is severely impaired. The small number of mature B cells that do develop in Lyn-deficient mice express normal levels of the transgenic BCR and lack expression of CD80 and CD86, suggesting they are not activated. In Lyn-deficient animals the presence of a Bcl-2 transgene leads to a dramatic increase in B cell numbers and restores T2 stage (IgM(hi) IgD(hi) CD21(hi) CD23(int)) and mature populations. In 3-83 lyn-/- Bcl-2 Tg mice, a population of lambda-positive cells that also express the 383 idiotype is evident, suggesting that in the absence of lyn isotype exclusion by the transgenic BCR is less efficient. The results indicate that Lyn plays a positive role in the selection and survival of mature B cells in addition to its previously documented negative role in tolerance and B cell activation.     

Elevated BCR signaling and decreased survival of Lyn-deficient transitional and follicular B cells

The Src-family tyrosine kinase Lyn negatively regulates BCR signaling and also myeloid cell activity. Mice deficient in Lyn have substantially decreased numbers of peripheral B cells, despite spontaneously producing IgG anti-DNA antibodies. Here, we examine the mechanism underlying the B-cell depletion in these mice. Lyn-deficient B cells were out-competed by WT B cells in mixed BM chimeras at two steps, at the T1 to T2 transitional maturation stage in the spleen and again between the T2 or T3 stage and the mature follicular B-cell population. Lyn-deficient T2 and follicular B cells expressed elevated levels of the pro-apoptotic factor Bim and deletion of Bim restored splenic B cells of Lyn-deficient mice to close to WT numbers. Lyn-deficient T2 and later stage B cells also had changes in cell surface phenotype consistent with increased in vivo BCR signaling. Similarly, an increased proportion of T2 and follicular B cells had elevated basal intracellular free calcium levels. Overall, these observations suggest that increased BCR signaling is responsible for increased death of weakly self-reactive Lyn-deficient B cells both at the T2 stage and additionally as these cells mature to follicular B cells.     

The culture of mouse bone marrow-derived mast cells (BMMC) and the anaphylactic release of chemical mediators]

Bone marrow cells from BALB/c, C3H/He and WBB6F1+/+ mice were cultured for 5 wks in the presence of the culture supernatant from prokoweed mitogen-stimulated spleen cells to assess and compare the degree of growth, proliferation and chemical mediator release of the mast cells (BMMC) derived from them. BMMC, which were positive to alcian blue staining, were found in the suspension cells on the culture of the bone marrow cells of either species of mice after 2 wk culture. The percentages of BMMC in the suspension cells were increased with time of culture, reaching more than 90% after 5 wks. No differences in the growth and proliferation rate among BMMC from these three species were observed. However, in regard to the amount of anaphylactic leukotriene (LT) and histamine release. BMMC from BALB/c mice were superior to those from other species. From the above results, subsequent experiments were executed with BMMC from BALB/c mice. There was no obvious difference in the releasability of anaphylactic mediators among BMMC obtained at any stages of the passage during 4-12 wk culture. On the other hand, although BMMC cultured for 4 and 5 wks well responded to Ca ionophore A23187 for these mediator release, those for 6 to 12 wks obviously deteriorated with prolongation of the culture. The time course of the anaphylactic release of immunoreactive (i-) LTB4, i-LTC4 and histamine from BMMC revealed that almost maximum release was reached at 10, 20 and 5 min, respectively, after antigen challenge. Several drugs including antiallergics and beta-stimulants had no effect on their release. From these results, it is suggested that present BMMC may be inadequate cells for evaluation of antiallergic drugs that can inhibit the anaphylactic mediator release, but may be useful for the research of the mechanism of the release because the cells likely release the mediators without occurrence of complicated subordinate reactions.     

Strain difference of murine bone marrow-derived mast cell functions

Mast cells play an important role for the induction and the expression of allergic responses. In this report, we studied the strain difference of bone marrow-derived murine mast cell (BMMC) functions in vitro. BMMC were induced by in vitro culture of bone marrow cells from BALB/c and C57BL/6 mice with interleukin (IL)-3 for 4 weeks, stimulated with immunoglobulin E antibody and antigen, and mediators and cytokines released in the culture supernatant were assayed. BMMC from C57BL/6 mice released a higher amount of granule-associated mediators, beta-hexosaminidase, and histamine than that from BALB/c mice. The expression of mRNA of histidine decarboxylase was higher in C57BL/6 mice. Conversely, the productions of newly synthesized mediators, prostaglandin D2 (PGD2), IL-6, and monocyte chemoattractant protein-1, and the mRNA expression of IL-5 were higher in BALB/c BMMC than C57BL/6 BMMC. Although mRNA and protein expression levels of cyclooxygenase-2 were equal in two strains, both expression levels of hematopoietic PGD synthase (hPGDS) were higher in BALB/c BMMC. Mast cells, freshly obtained from mice, also showed the same strain difference concerning the mediator release. These results indicate that the strain difference exists in mast cell functions in mice, and this difference can be considered to induce the susceptibility difference to allergic reactions in mouse strains.     

T lymphocytes from granulocyte colony-stimulating factor-/- mice produce large quantities of interferon-gamma in a chronic infection model

Little is known about the role of granulocyte colony-stimulating factor (G-CSF) in the response to chronic bacterial infections. To address this we infected G-CSF knock out (G-CSF-/-) mice with Mycobacterium avium. Infection was not exacerbated in G-CSF-/- mice despite a deficiency in the total bone marrow cells, colony-forming haemopoietic cells, granulocytes and monocyte precursors in the bone marrow. Peritoneal cells from G-CSF-/- produced less nitric oxide (NO) upon culture in vitro with antigen than did wild-type (WT) cells. Unexpectedly, T cells from infected G-CSF-/- mice were able to produce significantly more interferon-gamma (IFN-gamma) than the wild type (WT) controls. T cells from G-CSF-/- mice still produced more IFN-gamma even when in vitro NO production was inhibited, while enzyme-linked immunospot assay (ELISPOT) assays showed more IFN-gamma-producing cells in the G-CSF-/- mice. This was confirmed by intracellular cytokine staining (ICCS), which showed that there were more IFN-gamma producing T cells in vivo in the G-CSF-/- than the WT controls following M. avium infection. It is possible that a deficit of NO in vivo allows T cells to develop a higher IFN-gamma-producing phenotype. Thus we show a novel relationship between G-CSF and IFN-gamma production by T cells revealed in this chronic bacterial infection model.     

Lyn regulates inflammatory responses in Klebsiella pneumoniae infection via the p38/NF‐κB pathway

Klebsiella pneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is becoming increasingly multidrug resistant. However, the underlying molecular pathogenesis of this bacterium remains elusive, limiting the therapeutic options. Understanding the mechanism of its pathogenesis may facilitate the development of anti‐bacterial therapeutics. Here, we show that Lyn, a pleiotropic Src tyrosine kinase, is involved in host defense against Kp by regulating phagocytosis process and simultaneously downregulating inflammatory responses. Using acute infection mouse models, we observed that lyn^?/? mice were more susceptible to Kp with increased mortality and severe lung injury compared with WT mice. Kp infected‐lyn^?/? mice exhibited elevated inflammatory cytokines (IL‐6 and TNF‐α), and increased superoxide in the lung and other organs. In addition, the phosphorylation of p38 and NF‐κB p65 subunit increased markedly in response to Kp infection in lyn^?/? mice. We also demonstrated that the translocation of p65 from cytoplasm to nuclei increased in cultured murine lung epithelial cells by Lyn siRNA knockdown. Furthermore, lipid rafts clustered with activated Lyn and accumulated in the site of Kp invasion. Taken together, these findings revealed that Lyn may participate in host defense against Kp infection through the negative modulation of inflammatory cytokines.