Andrew W. Stadnyk

Cytokine production by epithelial cells.

The epithelium has long been regarded as a passive barrier intended to protect the underlying tissues. Many regulatory signals, including cytokines, that control epithelial cell proliferation and differentiation and cell function during inflammation were thought to be nonepithelial‐derived. Now there is a growing appreciation that epithelial cells provide some of the impetus for their own growth and differentiation, and may also regulate the function of other cells through the elaboration of certain cytokines. Moreover, because epithelial cells serve as the interface between the organism and environment, they are in a position to signal changes in the environment. It is now clear that epithelial cells respond to injury or infection with cytokine secretion. Various approaches to detecting cytokines in normal and diseased tissue have been undertaken during the past few years to establish cytokine synthesis by different epithelia. This review will examine these recent investigations in various general contexts of epithelial cell function.—Stadnyk, A. W. Cytokine production by epithelial cells. FASEB J. 8: 1041‐1047; 1994.

The functional significance behind expressing two IL–8 receptor types on PMN

PMN are critical to innate immunity and are fundamental to antibacterial defense. To localize to sites of infection, PMN possess receptors that detect chemoattractant stimuli elicited at the site, such as chemokines, complement split products, or bioactive lipids. Signaling through these receptors stimulates chemotaxis toward the site of infection but also activates a number of biochemical processes, with the result that PMN kill invading bacteria. PMN possess two receptors, CXCR1 and CXCR2, for the N–terminal ELR motif–containing CXC chemokines, although only two chemokine members bind both receptors and the remainder binding only CXCR2. This peculiar pattern in receptor specificity has drawn considerable interest and investigation into whether signaling through each receptor might impart unique properties on the PMN. Indeed, at first glance, CXCR1 and CXCR2 appear to be functionally redundant; however, there are differences. Considering these proinflammatory activities of activating PMN through chemokine receptors, there has been great interest in the possibility that blocking CXCR1 and CXCR2 on PMN will provide a therapeutic benefit. The literature examining CXCR1 and CXCR2 in PMN function during human and modeled diseases will be reviewed, asking whether the functional differences can be perceived based on alterations in the role PMN play in these processes.

Intestinal epithelial cells as a source of inflammatory cytokines and chemokines

The intestinal epithelium has long been known to provide nonspecific defences such as mucus, lysozyme and transport of secretory immunoglobulin via the polyimmunoglobulin receptor. In the past decade, the realization emerged that enterocytes secrete molecules (cytokines) that regulate inflammation. As the focus tightened on this new role as sentinel, so has the interest in enterocyte production of cytokines with chemoattractant properties for leukocytes - the chemokines. Neutrophils are a prominent feature of the cellular infiltrate in various inflammatory diseases, and early reports indicated that epithelial cells secrete neutrophil chemoattractants. More recently, it has been shown that the cells also secrete chemokines for monocytes and lymphocytes. Some of these chemokines appear to be important in the uninflamed intestine but become increased during disease. While a great deal of knowledge has been gained regarding the circumstances leading to chemokine production by epithelial cells, the application of this understanding to the treatment of human intestinal diseases is lacking. Closing this gap is necessary to take advantage of emerging therapies aimed at blocking chemokine function.

Oral Exposure to Alloantigen Generates Intragraft CD8+ Regulatory Cells

We have previously reported that oral administration of allogeneic rat spleen cells before kidney allotransplantation significantly prolongs graft survival. This prolongation was alloantigen specific and was associated with a decrease in graft-infiltrating cells (GIC) and an increase in transcription of IL-4 mRNA in the GIC. In this study increased splenic mixed lymphocyte responses from animals orally exposed to alloantigen before kidney transplantation suggested that the kidney allograft prolongation was not due to a masking of allorecognition, but to an immunomodulation of the immune response. We have assessed GIC T cell subsets on day 5 post-transplant and found decreased numbers of CD4+ T cells in fed animals compared with controls, but there was no change in CD8+ T cell numbers. The CD8+ GIC from fed animals transcribed substantial levels of perforin, granzyme, and Fas ligand mRNA, indicating the presence of active CTL. Direct CTL assays showed that the GIC from fed recipients exhibited higher allo-CTL activity than GIC from control unfed recipients. In addition, the CD8+ GIC exhibited high levels of IL-4 mRNA, suggesting Tc2-type regulatory cells. Prolonged graft survival in the face of active CTL and Tc2 cells suggests the presence of a CD8+ regulatory cell population in the allograft. To confirm this, cell transfer experiments were performed. Prolongation of graft survival was transferred from rats orally exposed to alloantigen to naive animals by transfer of CD8+ GIC. This is the first report that oral exposure to alloantigen prolongs kidney allograft survival by the generation of intragraft CD8+ regulatory cells.

Therapeutic Effect of Blocking CXCR2 on Neutrophil Recruitment and Dextran Sodium Sulfate-Induced Colitis

Dextran sodium sulfate (DSS)-induced colitis in mice is characterized by polymorphonuclear neutrophil (PMN) infiltration into the colonic mucosa and lumen. The mechanism by which this occurs is unclear. To begin to understand the mechanism, we determined the role of the PMN chemokine receptor, CXCR2, in DSS-induced colitis by using CXCR2(-/-) mice or by neutralizing CXCR2. DSS was administered through drinking water to CXCR2(-/-) and BALB/c mice for 5 days followed by regular water for 1 day. In the neutralization study, mice were injected with control serum or goat anti-CXCR2 antiserum. BALB/c mice receiving DSS and control serum-injected mice receiving DSS lost weight and showed considerable clinical illness. Histological observation revealed submucosal edema, PMN infiltration into the submucosa and mucosa, extensive crypt damage with abscesses, and ulceration. In contrast, both the CXCR2(-/-) and anti-CXCR2 antiserum-treated mice gained weight and had significantly lower symptom scores. Histology of these mice showed submucosal edema but relatively intact crypt architecture and very few ulcers. Significantly fewer PMNs were found in the mucosa in anti-CXCR2 anti-serum compared with control serum-injected inflamed mice, but no significant difference in eosinophil infiltration was observed between the groups. Our experiments identify a role for CXCR2 in DSS-induced colitis and suggest that antagonizing CXCR2 provides some therapeutic efficacy, possibly by impeding PMN recruitment into the mucosa. Antagonizing CXCR2 may form the basis for therapeutic drugs directed at controlling colitis.

Isolation and characterization of highly purified rat intestinal intraepithelial lymphocytes.

The study of intestinal intraepithelial lymphocytes (IEL) has been hindered by the difficulty of isolating a population of lymphocytes which is free of epithelial cell or lamina propria cell contaminants and representative of the in vivo population of IEL in both phenotype and function. We describe an improved technique for the extraction and purification of IEL from the proximal small intestine of the rat. This technique rapidly and reproducibly isolates 5-10 x 10(6) IEL/rat with 90-95% purity and viability without the use of enzymes which affect lymphocyte function. The resulting cell population, which is 75% alpha beta T cell receptor (TCR)+, 70% CD8+, and 33% CD4+ T cells, and only 5% B cells and 2% macrophages, is of suitable purity to allow for flow cytometric analysis of the entire population of cells without requiring gating on lymphocytes. IEL are comprised of a unique T cell repertoire in that 27% of cells co-express the CD4 and CD8 molecules, but only 11% of CD4+ cells co-express CD45RC. All CD4+ cells express the alpha beta TCR, but 9% of IEL are CD8+ CD4- alpha beta TCR-. The adhesion molecules alpha 4 integrin and L-selectin are expressed on 57% and less than 1% of IEL, respectively. The isolated IEL population contains mRNA for IL-1 alpha, IL-1 beta, IL-1R, IL-1RA, IL-2, IL-6R, IFN-gamma, TGF-alpha, TGF-beta 1, and TNF-alpha. Mesenteric lymph node cells (MLNC) were examined in parallel. This technique allows for the isolation of rat IEL appropriate for phenotypic analysis by flow cytometry and for cytokine analysis by reverse transcription/polymerase chain reaction.

Activation of NF-κB following detachment delays apoptosis in intestinal epithelial cells

We reported earlier that IL-1β, an NF-κB-regulated cytokine, was made by intestinal epithelial cells during detachment-induced apoptosis (anoikis) and that IL-1 was antiapoptotic for detached cells. Since surviving anoikis is a prerequisite for cancer progression and metastases, we are further exploring the link between anoikis and cytokines. Here we determined that multiple genes are expressed following detachment including a number of NF-κB-regulated products and therefore aimed to determine whether NF-κB signalling plays any role in regulating apoptosis. Using Western blotting, we detected that IκBα becomes phosphorylated immediately following detachment and that levels of phospho-IκBα peaked within 20 min. Phosphorylation of IκBα was followed by Rel A (p65) nuclear translocation. Increased NF-κB activity following detachment was confirmed using the detection of NF-κB-promoted luciferase gene expression delivered by adenovirus infection. Infection of cells with adenovirus expressing a super-repressor IκBα protein and pharmacological inhibitors of NF-κB resulted in the failure to phosphorylate IκBα, a more rapid activation of caspases and earlier apoptosis. We also detected that IκB kinase α (IKKα) and not IKKβ became phosphorylated following detachment. Since IKKα is activated by NF-κB-inducing kinase (NIK), we overexpressed native NIK using an adenovirus vector that resulted in enhanced phospho-IκBα and nuclear p65 in detached cells compared to control detached cells but did not result in a significantly greater number of cells surviving to 24 h. We conclude that detachment directly activates NF-κB, which, in addition to launching an inflammatory cytokine wave, contributes to a delay in apoptosis in intestinal epithelial cells.

Neutrophils migrate across intestinal epithelium using β2 integrin (CD11b/CD18)‐independent mechanisms

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.

E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin‐conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co‐crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

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.