Activation of RegIIIbeta/gamma and interferon gamma expression in the intestinal tract of SCID mice: an innate response to bacterial colonisation of the gut.

BACKGROUND AND AIMS: The mechanisms by which commensal bacteria provoke intestinal inflammation in animal models of inflammatory bowel disease (IBD) remain incompletely defined, leading to increasing interest in the innate immune response of the colonic mucosa to bacterial colonisation. METHODS: Using gene expression profiling of colonic RNA from C.B17.SCID germ free mice and those colonised with altered Schaedler's flora, we investigated the innate immune response to bacterial colonisation in vivo. The two most consistently induced gene groups were RegIIIbeta and gamma as well as interferon gamma (IFN-gamma) response genes. RESULTS: Using quantitative reverse transcription-polymerase chain reaction, we showed that RegIIIbeta, RegIIIgamma, and IFN-gamma were constitutively expressed in the colon of conventionally housed SCID mice compared with either germ free SCID or conventionally housed BALB/c mice. Induction of these genes was reproduced by chronic monoassociation of germ free SCID mice with either of two separate gut commensal bacterial species-segmented filamentous bacteria and Schaedler's Escherichia coli. The cellular source for IFN-gamma on monoassociation of SCID mice with Schaedler's E coli was localised to a subset of intraepithelial natural killer (IENK) cells that express asialo-GM1. In vivo IFN-gamma immunoneutralisation studies failed to demonstrate any alteration in RegIIIbeta or gamma expression. CONCLUSIONS: Thus bacterial colonisation of the colon independently activates two distinct innate immune cell types at the mucosal interface with the colonic lumen, intestinal epithelial cells, and IENK cells, a response that may be regulated by the adaptive immune system. These innate immune responses may play a role in the pathogenesis of colitis in SCID adoptive transfer models in mice and possibly in patients with IBD.

An open-label trial of the PPAR-gamma ligand rosiglitazone for active ulcerative colitis.

OBJECTIVES: Previous research has demonstrated that ligands for the gamma subtype of peroxisome proliferator-activated receptors (PPARs) reduce inflammation in two different murine models of colitis. This study was designed to examine the potential efficacy of rosiglitazone, a ligand for the gamma subtype of PPARs, as a therapy for active ulcerative colitis. METHODS: Fifteen patients with mild to moderately active ulcerative colitis despite therapy with 5-aminosalicylic acid compounds were enrolled in an open-label study of rosiglitazone (4 mg b.i.d. p.o.) for 12 wk. Thirteen of 15 patients were receiving concomitant therapy with corticosteroids and/or immunomodulator medications. Disease activity was measured with the Disease Activity Index. RESULTS: After 12 wk of therapy, four patients (27%) had achieved clinical remission, of whom three (20%) also had an endoscopic remission. Four additional patients (27%) had a clinical response without achieving remission. Two patients were hospitalized with worsened disease activity, and one patient was withdrawn for nephrotic syndrome. CONCLUSIONS: These data suggest that ligands for the gamma subtype of PPARs may represent a novel therapy for ulcerative colitis. A double blind, placebo-controlled, randomized trial is warranted.

High-level expression of I kappa B-beta in the surface epithelium of the colon: in vitro evidence for an immunomodulatory role.

The intestinal epithelium is spatially segregated into two compartments, one containing undifferentiated cells in a proliferative state and one with non-proliferative differentiated cells. Although this epithelium can produce many immunemodulating substances, emerging evidence suggests that the differentiated cell compartment is less immune responsive. Indeed, it is the differentiated cellular compartment that represents the interface between the highly antigenic luminal environment and the mucosal immune system. The NF-kappaB/rel family of transcriptional activators play a critical role in regulating the inflammatory response by activating a wide variety of immune-modulating genes. These transcription factors are maintained in an inactive state in the cytoplasmic compartment by interaction with inhibitory proteins of the IkappaB family. In this study we show by immunohistochemistry that IkappaB-beta is expressed at high levels specifically in the differentiated surface epithelium of the colonic mucosa. Using a naturally occurring compound found in the colon of vertebrates, butyrate, we provide evidence in an intestinal cell line that alteration of IkappaB-beta expression can modulate the transcriptional activation of the interleukin-8 (IL-8) gene by preventing the nuclear translocation of NF-kappaB proteins. Therefore, the expression of IkappaB-beta in the differentiated surface epithelium of the colon may help these cells act as an immunological barrier to prevent activation of the mucosal immune system.

A novel therapy for colitis utilizing PPAR-gamma ligands to inhibit the epithelial inflammatory response.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily originally shown to play a critical role in adipocyte differentiation and glucose homeostasis, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. Colonic epithelial cells, which express high levels of PPAR-gamma protein, have the ability to produce inflammatory cytokines that may play a role in inflammatory bowel disease (IBD). We report here that PPAR-gamma ligands dramatically attenuate cytokine gene expression in colon cancer cell lines by inhibiting the activation of nuclear factor-kappaB via an IkappaB-alpha-dependent mechanism. Moreover, thiazolidinedione ligands for PPAR-gamma markedly reduce colonic inflammation in a mouse model of IBD. These results suggest that colonic PPAR-gamma may be a therapeutic target in humans suffering from IBD.

Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues.

GAP-43 is an abundant protein in axonal growth cones of developing and regenerating neurons. We found that GAP-43 was enriched in detergent-resistant membranes (DRMs) isolated by Triton X-100 extraction from PC12 pheochromocytoma cells and could be detected in detergent-insoluble plasma membrane remnants after extraction of cells in situ. GAP-43 is palmitoylated at Cys-3 and Cys-4. Mutation of either Cys residue prevented association with DRMs. A hybrid protein containing the first 20 amino acid residues of GAP-43 fused to beta-galactosidase was targeted to DRMs even more efficiently than GAP-43 itself. We conclude that tandem palmitoylated Cys residues can target GAP-43 to DRMs, defining a new signal for DRM targeting. We propose that tandem or closely spaced saturated fatty acyl chains partition into domains or "rafts" in the liquid-ordered phase, or a phase with similar properties, in cell membranes. These rafts are isolated as DRMs after detergent extraction. The brain-specific heterotrimeric G protein Go, which may be regulated by GAP-43 in vitro, was also enriched in DRMs from PC12 cells. Targeting of GAP-43 to rafts may function to facilitate signaling through Go. In addition, raft association may aid in sorting of GAP-43 into axonally directed vesicles in the trans-Golgi network.

Evidence that the two C1q binding membrane proteins, gC1q-R and cC1q-R, associate to form a complex.

Two types of widely coexpressed, highly acidic, cell membrane binding proteins that display preferential domain specificity for C1q have been described: a 60-kDa calreticulin homologue, designated cC1q-R, that binds to the collagen-like "stalk" and a 33-kDa glycoprotein with affinity for the globular "heads" (gC1q-R). Although the two molecules are known to be coexpressed on all cell types examined to date and often coelute during purification, there is no direct evidence showing that they associate with each other either on the membrane or when examined in a purified system. In this report we present the first evidence that 1) biotinylated cC1q-R binds to recombinant as well as native gC1q-R, as assessed by solid phase ELISA; 2) binding sites for cC1q-R are located within N-terminal residues 76 through 93 of the mature form of gC1q-R and within residues 204 through 218; 3) this interaction is inhibited by two mAbs, 60.11 and 46.23, that recognize primarily epitopes within the N terminus of gC1q-R corresponding to residues 74 through 96 and by mAb 74.5.2 that recognizes epitopes within residues 204 through 218; and 4) biotinylated cC1q-R binds to microtiter-fixed Raji and K562 cells, and this interaction is inhibited by mAb 60.11. Furthermore, coimmunoprecipitation analysis of Raji cell membranes with anti-gC1q-R mAbs showed the presence of cC1q-R in addition to gC1q-R. Taken together, the evidence suggests that cC1q-R is able to form a complex with gC1q-R and may associate with gC1q-R on the cell surface.

The C1q-binding cell membrane proteins cC1q-R and gC1q-R are released from activated cells: subcellular distribution and immunochemical characterization.

Two types of widely coexpressed cell surface C1q-binding proteins (C1q-R): a 60-kDa calreticulin-homolog which binds to the collagen-like "stalk" of C1q and a 33-kDa protein with affinity for the globular "heads" of the molecule, have been described. In this report, we show that the two molecules are also secreted by Raji cells and peripheral blood lymphocytes and can be isolated in soluble form from serum-free culture supernatant by HPLC purification using a Mono-Q column. The two purified soluble proteins had immunochemical and physical characteristics similar to their membrane counterparts in that both bound to intact C1q and to their respective C1q ligands, cC1q and gC1q. In addition, N-terminal amino acid sequence analyses of the soluble cC1q-R and gC1q-R were found to be identical to the reported sequences of the respective membrane-isolated proteins. Ligand blot analyses using biotinylated membrane or soluble cC1q-R and gC1q-R showed that both bind to the denatured and nondenatured A-chain and moderately to the C-chain of C1q. Moreover, like their membrane counterparts, the soluble proteins were found to inhibit serum C1q hemolytic activity. Although cC1q-R was released when both peripheral blood lymphocytes and Raji cells were incubated in phosphate-buffered saline for 1 hr under tissue culture conditions, gC1q-R was releasable only from Raji cells, suggesting that perhaps activation or transformation leading to immortalization is required for gC1q-R release. Subcellular fractionation of Raji cells and analyses by enzyme-linked immunosorbent assay and Western blotting showed that the two molecules are present in the cytosolic fractions as well as on the membrane. The data suggest that soluble forms of both C1q-binding molecules are released from cells and that these molecules may play important roles in vivo as regulators of complement activation.

Effect of 2',3'-dideoxycytidine on oxidative phosphorylation in the PC12 cell, a neuronal model.

Peripheral neuropathy induced by 2',3'-dideoxycytidine (ddC) could result from the previously shown inhibition of mtDNA replication by the action of ddC on the mitochondrial enzyme DNA polymerase gamma. Such inhibition would be expected to impair oxidative phosphorylation, and this was demonstrated in the present study for the PC12 cell, a model of a peripheral neuron. The dramatic rise in lactate formation upon exposure of the cell to ddC indicated that increased glycolysis was needed to produce ATP. A concomitant rise in O2 uptake indicated that oxidative phosphorylation had become uncoupled. When tested in a standard respiratory control system (isolated rat liver mitochondria), however, we found ddC not to be an uncoupler. Rather, the uncoupling most likely resulted from the failure of synthesis of one or more mitochondrial gene products necessary for oxidative phosphorylation. We also observed an important distinction between the manner in which ddC and 3'-azido-3'-deoxythymidine (AZT) act. ddC-exerted inhibition of oxidative phosphorylation was delayed for several days. This is consistent with the inhibition occurring indirectly, most likely as a result of the prior destruction of the mitochondrial genome, which encodes many of the components of the oxidative phosphorylation system. In contrast, we have shown previously that although AZT also impairs replication of the mitochondrial genome (in the Friend murine erythroleukemic cell), it also attacks directly an additional primary target leading to impairment of oxidative phosphorylation; its initial inhibition of this process is immediate, not occurring via inhibition of mitochondrial DNA replication.

Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain.

Recent cloning of auxiliary subunits associated with voltage-gated ion channels and their subsequent coexpression with the channel forming alpha-subunits has revealed that the expression level, gating and conductance properties of the expressed channels can be profoundly affected by the presence of an auxiliary subunit polypeptide. In the present study, we raised antibodies against the beta-subunit associated with the bovine dendrotoxin sensitive K(+)-channel complex and used these antibodies to characterize the related beta-subunit polypeptides in rat brain. The anti-beta-subunit antibodies displayed a specific reaction on immunoblots of rat brain membranes with a major 38 kDa polypeptide, and a minor 41 kDa polypeptide, which correspond closely to the predicted sizes of the Kv beta 2 and Kv beta 1 beta-subunit polypeptides, respectively, recently cloned from rat brain. Reciprocal coimmunoprecipitation experiments revealed that the beta-subunit polypeptides are associated with Kv1.2 and Kv1.4, but not Kv2.1, alpha-subunits. Immunohistochemical staining revealed that the beta-subunit polypeptides were widely distributed in adult rat brain. Moreover, the cellular distribution of beta-subunit immunoreactivity corresponded closely with immunoreactivity for Kv1.2, and to a lesser extent Kv1.4, but not with Kv2.1. These results suggest that neuronal mechanisms may exist to direct the selective interaction of K+ channel alpha- and beta-subunit polypeptides, and that the properties of K+ channels in specific subcellular domains may be regulated by the formation of heteromultimeric K+ channel complexes containing specific combinations of alpha- and beta-subunits.

Cellular targets of 3'-azido-3'-deoxythymidine: an early (non-delayed) effect on oxidative phosphorylation.

Previous results demonstrated that incubation of the Friend murine erythroleukemic cell with 5 microM AZT for several days leads to a decrease in the rate of cell growth, inhibition of mtDNA replication, reduction of mtDNA per cell and per mitochondrion, and an increase in mitochondria per cell. As shown here, such treatment also leads to changes in lactate and ATP synthesis and in O2 uptake, suggesting impairment of oxidative phosphorylation. Direct measurement of ATP synthesis in mitochondria isolated from AZT-treated cells confirmed this view. The most significant new finding in this paper, however, is that in addition to these delayed effects of AZT, similar but very rapidly appearing effects on oxidative phosphorylation were noted, with changes observed in the above parameters including mitochondrial proliferation. Some of these occurred as early as 3 hr, only 7% of the doubling time, after exposure of the cells to 5 microM AZT, a period too short for initiation of appreciable mtDNA-mediated effects. Studies on isolated mitochondria provided no evidence of the identity of the immediate target of AZT: AZT does not act as an uncoupler or inhibitor of respiratory control, and previous results failed to implicate adenylate kinase. We have also begun to address the question of the mechanism of AZT-induced mitochondrial proliferation. Initial experiments showed that AZT inhibited synthesis of total cytosolic protein but stimulated synthesis of those proteins imported into mitochondria from the cytoplasm. We also report that aminothymidine, a catabolite of AZT in liver capable of inhibiting cell growth, was not generated by Friend cells.

Mechanisms of toxicity of 3'-azido-3'-deoxythymidine. Its interaction with adenylate kinase.

Recent experiments from our laboratory have indicated that the inhibitory effect of 3'-azido-3'-deoxythymidine (AZT) on oxidative phosphorylation may occur directly, in addition to being brought about by its inhibition of mtDNA replication. We report here studies on the effect of AZT on adenylate kinase, an enzyme crucial to oxidative phosphorylation. AZT decreased the aromatic residues fluorescence of rabbit muscle adenylate kinase, indicating binding of AZT to the enzyme. Of three other enzymes studied as controls, AZT bound only to those that possessed ATP/ADP binding sites. Up to concentrations of 15 microM, AZT was a more potent effector of fluorescence quenching than were ATP, ADP, AMP, and the AZT control, deoxythymidine. AZT strongly inhibited adenylate kinase in the direction of ATP synthesis (Ki, 8 microM), the inhibition being of the partial competitive type, whereas deoxythymidine inhibition, also partially competitive, was much weaker (Ki, 90 microM). When measured in the direction of ADP synthesis, AZT failed to demonstrate any inhibition at concentrations up to 10 microM. Experiments on isolated intact rat liver mitochondria with the enzyme activity measured in both directions confirmed the isolated enzyme results. Respiratory control by these mitochondria was not affected by AZT. The finding of AZT affinity for ATP/ADP binding sites may open new avenues of approach to the study of AZT toxicity.

Anti-human immunodeficiency virus type 1 therapy and peripheral neuropathy: prevention of 2',3'-dideoxycytidine toxicity in PC12 cells, a neuronal model, by uridine and pyruvate.

A strategy for preventing or delaying the peripheral neuropathy induced by 2',3'-dideoxycytidine (ddC) therapy in patients with acquired immunodeficiency syndrome was suggested by findings, in two laboratories, that cultured avian and mammalian cells devoid of mitochondrial DNA continue to replicate at virtually normal rates, provided that the medium is supplemented with uridine and pyruvate. Inasmuch as it is likely that a depletion of mitochondrial DNA also takes place in neuronal cells exposed to ddC, we used PC12 cells, the neuronal model we have reported on previously, in an attempt to rescue these cells from the deleterious effects of ddC. We first show, using undifferentiated PC12 cells, that DNA replication is impaired in mitochondria isolated from cells grown in the presence of ddC. Then, using growth rate as a criterion of the well-being of the cells, we show that the addition of uridine and pyruvate to uninduced cells growing in the presence of ddC results in an average rescue efficiency of 51%, based on the uridine/pyruvate-treated control. This value increases considerably at substantially higher concentrations of uridine alone. Rescue efficiencies of differentiated cells, which do not proliferate, were assessed using neurite outgrowth and neurite survival as criteria. Here the rescue efficiency is 56%, based on the uridine/pyruvate-treated control. In addition, uridine and pyruvate prolong the viability of ddC-treated cells and maintain their healthy appearance; without these compounds, the ddC-treated cells have an abnormal morphology and die off quite rapidly.