SUMMARY Recruitment of polymorphonuclear leucocytes (PMN) across the intestinal epithelium is dependent on specific adhesion molecules and chemoattractants diffusing from the intestinal lumen. The present understanding is that in response to fMLP, PMN migration across a T84 colon carcinoma monolayer is dependent on the β2 integrin, Mac‐1 (CD11b/CD18). To further understand PMN transepithelial migration, we sought to determine whether migration to C5a, IL‐8 and LTB4 was similarly Mac‐1‐, or even CD18‐dependent. T84 epithelial cell monolayers growing on Transwell filters were used in combination with radiolabelled peripheral blood PMN. The number of migrated PMN was established by the amount of radioactivity recovered from the well after the migration period. Monoclonal antibodies were used to block integrin function. Whereas essentially all migration to fMLP across T84 monolayers was prevented by anti‐CD18 antibody, significant migration to C5a, IL‐8 or LTB4 persisted despite anti‐CD18 antibody, indicating PMN are capable of β2 integrin‐independent transepithelial migration. An antibody to  CD11b  but  not  CD11a  blocked  migration  to  an  extent  similar  as  with anti‐CD18.  CD18‐independent  PMN migration to C5a occurred only in the basolateral‐to‐apical direction across epithelial cells.  Co‐stimulation of PMN with C5a and fMLP or IL‐8 plus LTB4 and fMLP still resulted in CD18‐independent migration. Thus CD18 use during PMN migration across this model epithelium is a function of the chemoattractant inducing migration. The finding of CD18‐independent migration mechanisms needs to be considered when developing antiadhesion molecule strategies to reduce or reverse intestinal inflammation.

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Summary During active intestinal inflammation granulocytes accumulate in the lumen of the gut where they damage the epithelium through the release of various products such as reactive oxygen species and proteolytic enzymes. Previously, using function blocking monoclonal antibodies, we showed that neutrophil migration across intestinal epithelial monolayers in response to various chemoattractants was partially β2 integrin Mac‐1 (CD11b/CD18)‐independent. Here, we show that treating neutrophils with intact monoclonal antibody (mAb) to CD18 activates the cells to express more CD11b. Thus our goal now was to determine whether neutrophil Mac‐1‐independent transepithelial migration proceeds independently of prior cell activation through Mac‐1. We took two approaches, one using blocking Fab′ fragments of mAb to CD18 and the second was to develop a neutrophil differentiated HL‐60 cell line which is Mac‐1 deficient to further study neutrophil/epithelial cell interaction. Anti‐CD18 Fab′ minimally activated neutrophils but inhibited approximately 75% of transepithelial migration to fMLP while having a minimal effect (≤25% inhibition) on the migration to C5a. Upon incubation with dimethylsulphoxide, HL‐60 cells differentiated and up‐regulated CD11b expression and migrated to C5a and n‐formyl methionyl leucyl phenylalanine in a similar manner to peripheral blood neutrophils. In contrast, CD11b expression was minimal on HL‐60 cells differentiated with dibutytyl cAMP to a neutrophil‐like phenotype. These cells, however, readily migrated across both intestinal and lung epithelial monolayers in response to C5a. We conclude that Mac‐1‐independent transepithelial migration does not require prior activation of cells via Mac‐1 ligation because HL‐60 cells lacking Mac‐1 (CD11b/CD18) expression migrate effectively. HL‐60 cells differentiated with dbcAMP should greatly assist in the search for the Mac‐1‐independent ligands for neutrophil migration across epithelium.

In Crohn's disease and ulcerative colitis patients, the numbers of neutrophils recovered from stool directly correlates with the severity of disease, implying that neutrophils in the lumen contribute to the tissue destruction; therefore, it is important to understand the mechanisms behind transintestinal epithelial migration. Neutrophil transintestinal epithelial migration to fMLP is appreciated to be CD11b/CD18 integrin (Mac-1)-dependent, while we recently reported that migration to C5a is Mac-1-independent. Here, we investigated whether phospholipase D (PLD), a signaling molecule linked to chemoattractant activation of neutrophils, is necessary for both Mac-1-dependent and Mac-1-independent migration. Both fMLP and C5a increased neutrophil expression of the Mac-1 activation epitope, indicating PLD was activated. This up-regulation was dose-dependently prevented by incubation of neutrophils in 1-butanol, an inhibitor of PLD activity. Despite this effect on Mac-1, 1-butanol did not prevent neutrophil migration across acellular filters. Incubation in 1-butanol did inhibit fMLP but not C5a-mediated migration across intestinal epithelial cell monolayers, showing that transepithelial migration to fMLP but not C5a is dependent on PLD. The addition of phosphatidic acid, a reaction product of PLD, partially restored fMLP-mediated transepithelial migration in the presence of 1-butanol but not the migration of Mac-1-deficient neutrophil-differentiated HL-60 cells. Thus PLD control over expression of the Mac-1 activation epitope is critical for neutrophil migration to fMLP but not C5a. Moreover, as PLD controls other neutrophil functions, such as the oxidative response, degranulation, and protease release, we could exclude these functions as being important in neutrophil transepithelial migration to C5a.

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TRAF6 (tumor necrosis factor-associated factor 6) is an essential adaptor downstream from the tumor necrosis factor (TNF) receptor and Toll-like receptor superfamily members. This molecule is critical for dendritic cell maturation and T cell homeostasis. Here we show that TRAF6 is important in high affinity IgE receptor, Fc⑀RI-mediated mast cell activation. In contrast to dendritic cells and T cells, TRAF6-deficient mast cells matured normally and showed normal IgE-dependent degranulation. Importantly, TRAF6-deficient mast cells showed impaired production of cytokine interleukin-6, CCL-9, interleukin-13, and TNF following Fc⑀RI aggregation. Chromatin immunoprecipitation assay showed decreased NF-B p65 binding to CCL-9 and TNF promoters in TRAF6-deficient mast cells. Antigen and IgE-induced IB phosphorylation and NF-B p65 translocation to the nucleus were diminished in TRAF6-deficient mast cells. NF-B luciferase activity in response to antigen and IgE stimulation was severely impaired in TRAF6-deficient mast cells. In addition, antigen and IgE-induced phosphorylation of mitogen-activated protein kinase p38 and JNK, but not ERK1/2, was significantly reduced in TRAF6-deficient mast cells. These results identified TRAF6 as an important signal transducer in Fc⑀RI-mediated signaling in mast cells. Our findings implicate TRAF6 as a new adaptor/regulator molecule for allergen-mediated inflammation in allergy.

The presence of eosinophils in the lung is often regarded as a defining feature of asthma. On allergen stimulation, numbers of eosinophils and their progenitors are increased in both the bone marrow and lungs. Eosinophil progenitors provide an ongoing supply of mature eosinophils. Here, we report that deficiency in the regulator of calcineurin 1 gene (Rcan1) leads to a near-complete absence of eosinophilia in ovalbumin-induced allergic asthma in mice. In the absence of Rcan1, bone marrow cells produce significantly fewer eosinophils in vivo and in vitro on interleukin-5 stimulation. Importantly, eosinophil progenitor populations are significantly reduced in both naïve and ovalbumin-challenged Rcan1 mice. Bone marrow cells from Rcan1؊/؊ mice are capable of developing into fully mature eosinophils, suggesting that Rcan1 is required for eosinophil progenitor production but may not be necessary for eosinophil maturation. Thus, Rcan1 represents a novel contributor in the development of eosinophilia in allergic asthma through regulation of eosinophil progenitor production.

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