Tong-Jun Lin

Human Mast Cells Transmigrate Through Human Umbilical Vein Endothelial Monolayers and Selectively Produce IL-8 in Response to Stromal Cell-Derived Factor-1α

Mature mast cells are generally considered to be less mobile cells residing within tissue sites. However, mast cell numbers are known to increase in the context of inflammation, and mast cells are recognized to be important in regulating local neutrophil infiltration. CXC chemokines may play a critical role in this process. In this study two human mast cell-like lines, HMC-1 and KU812, and human cord blood-derived primary cultured mast cells were employed to examine role of stromal cell-derived factor-1 (SDF-1) in regulating mast cell migration and mediator production. It was demonstrated that human mast cells constitutively express mRNA and protein for CXCR4. Stimulation of human mast cells with SDF-1, the only known ligand for CXCR4, induced a significant increase in intracellular calcium levels. In vitro, SDF-1α mediated dose-dependent migration of human cord blood-derived mast cells and HMC-1 cells across HUVEC monolayers. Although SDF-1α did not induce mast cell degranulation, it selectively stimulated production of the neutrophil chemoattractant IL-8 without affecting TNF-α, IL-1β, IL-6, GM-CSF, IFN-γ, or RANTES production, providing further evidence of the selective modulation of mast cell function by this chemokine. These findings provide a novel, SDF-1-dependent mechanism for mast cell transendothelial migration and functional regulation, which may have important implications for the local regulation of mast cells in disease.

A Role of Toll-IL-1 Receptor Domain-Containing Adaptor-Inducing IFN-β in the Host Response to Pseudomonas aeruginosa Lung Infection in Mice

Toll-IL-1R domain-containing adaptor-inducing IFN-β (TRIF) is an adaptor molecule that mediates a distinct TLR signaling pathway. Roles of TRIF in the host defense have been primarily associated with virus infections owing to the induction of IFN-αβ. In this study, we investigated a role of TRIF in Pseudomonas aeruginosa infection. In vitro, TRIF-deficient mouse alveolar and peritoneal macrophages showed a complete inhibition of RANTES (CCL5) production, severely impaired TNF and KC (CXCL1) production, and reduced NF-κB activation in response to P. aeruginosa stimulation. In vivo, TRIF-deficient mice showed a complete inhibition of RANTES production, a severely impaired TNF and KC production, and an efficient MIP-2 and IL-1β production in the lung following P. aeruginosa infection. This outcome was associated with a delayed recruitment of neutrophils into the airways. These results suggest that TRIF mediates a distinct cytokine/chemokine profile in response to P. aeruginosa infection. P. aeruginosa-induced RANTES production is completely dependent on TRIF pathway in mice. Importantly, TRIF deficiency leads to impaired clearance of P. aeruginosa from the lung during the initial 24–48 h of infection. Thus, TRIF represents a novel mechanism involved in the development of host response to P. aeruginosa infection.

Mast Cells Down-Regulate CD4+CD25+ T Regulatory Cell Suppressor Function via Histamine H1 Receptor Interaction

Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (Treg) cell function. In this study, we show for the first time that the capacity of murine CD4+CD25+ Treg cells to suppress in vitro proliferation by CD4+CD25− T responder (Tresp) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcεR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited Treg cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored Treg cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of Treg cells with loratadine, but not famotidine, rescued Treg cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited Treg cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both Treg cells and Tresp cells expressed H1 receptors. Exposure to histamine caused Treg cells to express lower levels of CD25 and the Treg cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited Treg cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in Treg cell suppressor function, thereby enhancing the development of protective immunity.

Phosphatase inhibition potentiates IL-6 production by mast cells in response to FcεRI-mediated activation: involvement of p38 MAPK

Mast cells are crucial effector cells in the immune response through mediator secretion and release of cytokines. A coordinated balance between protein kinases and phosphatases plays an essential role in the regulation of mast cell mediator secretion. We have previously shown that treatment of mast cells with okadaic acid (OA), a protein phosphatase 2A (PP2A) inhibitor, results in a dose‐dependent increase in interleukin (IL)‐6 production. We show here for the first time a synergism between OA and immunoglobulin E (IgE)‐mediated IL‐6 secretion by murine bone marrow‐derived mast cells (BMMC). Selective p38 mitogen‐activated protein kinase (p38 MAPK) inhibition reduces OA and IgE‐mediated IL‐6 production. Regulation of p38 MAPK by PP2A was demonstrated, as OA treatment caused a dose‐dependent increase in p38 MAPK phosphorylation. Antigen‐mediated activation of murine mast cells also resulted in an increase in p38 MAPK phosphorylation, which was potentiated by cotreatment of the cells with OA. Lastly, in two mast cell lines (human mast cell‐1 5C6 and murine MC/9) and primary‐cultured murine BMMC, we show by coimmunoprecipitation an interaction between p38 MAPK and PP2A. These data support a role for PP2A through interaction with p38 MAPK in the regulation of IgE‐dependent mast cell activation.

Inhibition of IKK down-regulates antigen + IgE-induced TNF production by mast cells: a role for the IKK-IκB-NF-κB pathway in IgE-dependent mast cell activation

Mast cells (MC) are major effector cells for allergic diseases. Cross‐linking of immunoglobulin E (IgE) and its high‐affinity receptor, FcɛRI, by antigen initiates a cascade of signaling events leading to nuclear factor (NF)‐κB activation and tumor necrosis factor (TNF) production. Here, we demonstrated that inhibition of inhibitor of κB (IκB) kinase (IKK) by a peptide IKK inhibitor or by four individual chemical IKK inhibitors including 15‐deoxy‐prostaglandin J2, BMS‐345541, SC‐514, or sulindac significantly blocked IgE + trinitrophenyl (TNP)‐induced TNF production by mouse bone marrow‐derived MC (BMMC). Moreover, IgE + TNP induced a rapid phosphorylation of IKKα but not IKKβ in BMMC. IgE + TNP‐induced phosphorylation of IKKα was accompanied with phosphorylation and degradation of IκBα, subsequent NF‐κB activation, and TNF production. Inhibition of IKK by sulindac decreased IKKα phosphorylation, IκBα phosphorylation and degradation, NF‐κB activation, and TNF production by BMMC. It is interesting that IgE + TNP stimulation also induced a prominent synthesis of IKKα and IκBα. Inhibition of NF‐κB activity by pyrrolidine dithiocarbomate (PDTC) blocked IgE + TNP‐induced IκBα synthesis. NF‐κB activity and TNF production were also inhibited when PDTC was used even after IgE + TNP stimulation, suggesting a potential role for the newly synthesized IκBα in MC activation. In addition, IgE + TNP‐induced IKKα and IκBα phosphorylation was inhibited by a protein kinase C (PKC) inhibitor Ro 31‐8220. Taken together, our results support a role for the IKK‐IκB‐NF‐κB pathway, which likely involves PKC in IgE‐dependent TNF production by MC. Thus, IKK may serve as a new target for the regulation of MC function in allergy.

Selective Early Production of CCL20, or Macrophage Inflammatory Protein 3α, by Human Mast Cells in Response to Pseudomonas aeruginosa

ABSTRACT Mast cells are important as sentinel cells in host defense against bacterial infection. Much of their effectiveness depends upon recruiting other immune cells; however, little is known about the mechanisms of this response. CCL20, also known as macrophage inflammatory protein-3α (MIP-3α), Exodus, and LARC, is a chemokine known to be a potent chemoattractant for immature dendritic cells and T cells. In this study, we examined the human mast cell production of both CCL20 and granulocyte-macrophage colony-stimulating factor (GM-CSF), a critical cytokine for innate immune responses in the lung, in response to Pseudomonas aeruginosa. Reverse transcription-PCR and Western blot analysis demonstrated that the human mast cells (HMC-1) express CCL20 mRNA and are able to produce a significant amount (32.4 ng/ml) of CCL20 protein following stimulation by calcium ionophore and phorbol myristate acetate. Importantly, P. aeruginosa potently stimulated CCL20 production in human cord blood-derived mast cells (CBMC), with production peaking at 6 h after stimulation. This time course of expression was distinct from that of GM-CSF, which peaked after 24 to 48 h. Significant CCL20 production did not occur following immunoglobulin E-mediated activation of CBMC under conditions which induced a substantial GM-CSF response. Interestingly, the CCL20 response of mast cells to P. aeruginosa was relatively resistant to inhibition by the corticosteroid dexamethasone, interleukin-10, or cyclosporine, while GM-CSF production was potently inhibited. However, P. aeruginosa-induced CCL20 production was blocked by the protein kinase C (PKC) inhibitor Ro 31-8220 and a PKC pseudosubstrate. These results support a role for human mast cells in the initiation of immune responses to P. aeruginosa infection.

Autophagy Enhances Bacterial Clearance during P. aeruginosa Lung Infection

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.

Zebrafish mast cells possess an FcɛRI-like receptor and participate in innate and adaptive immune responses

We previously identified a zebrafish mast cell (MC) lineage and now aim to determine if these cells function analogously in innate and adaptive immunity like their mammalian counterparts. Intraperitoneal (IP) injection of compound 48/80 or live Aeromonas salmonicida resulted in significant MC degranulation evident histologically and by increased plasma tryptase compared with saline-injected controls (p=0.0006, 0.005, respectively). Pre-treatment with ketotifen abrogated these responses (p=0.0004, 0.005, respectively). Cross-reactivity was observed in zebrafish to anti-human high-affinity IgE receptor gamma (FcɛRIγ) and IgE heavy chain-directed antibodies. Whole mount in situ hybridization on 7-day embryos demonstrated co-localization of cpa5, a MC-specific marker, with myd88, a toll-like receptor adaptor, and zebrafish FcɛRI subunit homologs. Zebrafish injected IP with matched dinitrophenyl-sensitized mouse (anti-DNP) IgE and DNP-BSA or trinitrophenyl-sensitized mouse (anti-TNP) IgE and TNP-BSA demonstrated increased plasma tryptase compared with mismatched controls (p=0.03, 0.010, respectively). These results confirm functional conservation and validate the zebrafish model as an in vivo screening tool for novel MC modulating agents.

IFN regulatory factor 3 contributes to the host response during Pseudomonas aeruginosa lung infection in mice.

Pseudomonas aeruginosa is a major opportunistic pathogen. However, host defense mechanisms involved in P. aeruginosa lung infection remain incompletely defined. The transcription factor IFN regulatory factor 3 (IRF3) is primarily associated with host defense against viral infections, and a role of IRF3 in P. aeruginosa infection has not been reported previously. In this study, we showed that IRF3 deficiency led to impaired clearance of P. aeruginosa from the lungs of infected mice. P. aeruginosa infection induced IRF3 translocation to the nucleus, activation of IFN-stimulated response elements (ISRE), and production of IFN-β, suggesting that P. aeruginosa activates the IRF3–ISRE–IFN pathway. In vitro, macrophages from IRF3-deficient mice showed complete inhibition of CCL5 (RANTES) and CXCL10 (IP-10) production, partial inhibition of TNF, but no effect on CXCL2 (MIP-2) or CXCL1 (keratinocyte-derived chemokine) in response to P. aeruginosa stimulation. In vivo, IRF3-deficient mice showed complete inhibition of CCL5 production and partial or no effects on production of other cytokines and chemokines in the bronchoalveolar lavage fluids and lung tissues. Profiling of immune cells in the airways revealed that neutrophil and macrophage recruitment into the airspace was reduced, whereas B cell, T cell, NK cell, and NKT cell infiltration was unaffected in IRF3-deficient mice following P. aeruginosa lung infection. These data suggest that IRF3 regulates a distinct profile of cytokines and chemokines and selectively modulates neutrophil and macrophage recruitment during P. aeruginosa infection. Thus, IRF3 is an integral component in the host defense against P. aeruginosa lung infection.

Toll-like receptors in the host defense against Pseudomonas aeruginosa respiratory infection and cystic fibrosis

TLRs function in innate immunity by detecting conserved structures present in bacteria, viruses, and fungi. Although TLRs do not necessarily distinguish pathogenic organisms from commensals, in the context of compromised innate immunity and combined with pathogensˈ effector molecules, TLRs drive the host response to the organism. This review will discuss the evidence and role(s) of TLRs in the response to the opportunistic bacterial pathogen, Pseudomonas aeruginosa, as it relates to respiratory infection and CF, in which innate immune mechanisms are indeed compromised. Outer membrane lipoproteins, LPS, flagellin, and nucleic acids all serve as ligands for TLR2, ‐4, ‐5, and ‐9, respectively. These TLRs and their respective downstream effector molecules have proven critical to the host response to P. aeruginosa, although the protective effects of TLRs may be impaired and in some cases, enhanced in the CF patient, contributing to the particular susceptibility of individuals with this disease to P. aeruginosa infection.