Neutrophil migration into indomethacin induced rat small intestinal injury is CD11a/CD18 and CD11b/CD18 co-dependent.

BACKGROUND: Neutrophils may exacerbate intestinal inflammatory diseases through secretion of proteolytic enzymes and reactive oxygen and nitrogen intermediates. AIMS: To define the mechanisms involved in neutrophil infiltration into the non-steroidal anti-inflammatory disease inflamed intestine to develop strategies to regulate this process. METHODS: The small intestinal epithelium of (15 mg/kg) indomethacin treated rats was examined for cytokine mRNA. The kinetics of neutrophil accumulation into the gastrointestinal tract (including lumen contents) of inflamed rats was determined using radiolabelled (111In) neutrophils injected intravenously followed by a three hour migration period. To determine which adhesion molecules were critical for migration, rats were also injected with function blocking monoclonal antibodies to the beta2 (CD11/CD18) integrins. RESULTS: Interleukin 1beta, interleukin 1 receptor II, tumour necrosis factor alpha, and monocyte inflammatory peptide 2 but not monocyte chemoattractant protein 1 mRNA were detected in the epithelium within hours of indomethacin injection. Neutrophils were detectable in the small intestine and intestinal lumen by six hours and continued to accumulate until 48 hours post indomethacin injection. Neutrophil accumulation in the intestine was essentially blocked by anti-CD18, and partially blocked by either anti-CD11a or CD11b antibody treatment. Migration into the intestinal lumen was reduced by anti-CD11b. CONCLUSIONS: The small intestinal epithelium acts as one source of cytokines with properties important in the recruitment of neutrophils. In turn, neutrophil migration into the indomethacin inflamed small intestine is mediated by CD11a/CD18 and CD11b/CD18.

Neutrophil migration stimulates rat intestinal epithelial cell cytokine expression during helminth infection.

We are interested in understanding the role of epithelial cells during inflammation, and we previously reported that rat small intestinal epithelial cells express interleukin-1beta (IL-1beta) during infection by Trichinella spiralis. We now report that the epithelium also produces the potent neutrophil chemotactic factor, macrophage inflammatory protein-2 (MIP-2), and an IL-1 antagonist: the type II IL-1 receptor. Consequently we investigated the pattern of neutrophil infiltration into the infected intestine, which closely paralleled the epithelial cytokine expression. Speculating that neutrophil infiltration may provoke epithelial cytokine expression, neutrophil migration into the infected gut was reduced by depleting circulating cells through the use of a specific antibody, or by preventing migration through the use of a function-blocking anti-CD18 monoclonal antibody. Either treatment reduced the number of neutrophils recoverable from the small intestinal epithelium and was paralleled by reduced mRNA levels for epithelial cytokines. These results demonstrate that neutrophil infiltration of the small intestinal epithelium contributes to the stimulation of epithelial cell cytokines.

The Saccharomyces cerevisiae SPT7 gene encodes a very acidic protein important for transcription in vivo.

Mutations in the SPT7 gene of Saccharomyces cerevisiae originally were identified as suppressors of Ty and delta insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Other genes that have been identified in mutant hunts of this type have been shown to play a role in transcription. In this work we show that SPT7 is also important for proper transcription in vivo. We have cloned and sequenced the SPT7 gene and have shown that it encodes a large, acidic protein that is localized to the nucleus. The SPT7 protein contains a bromodomain sequence; a deletion that removes the bromodomain from the SPT7 protein causes no detectable mutant phenotype. Strains that contain an spt7 null mutation are viable but grow very slowly and have transcriptional defects at many loci including insertion mutations, Ty elements, the INO1 gene and the MFA1 gene. These transcriptional defects and other mutant phenotypes are similar to those caused by certain mutations in SPT15, which encodes the TATA binding protein (TBP). The similarity of the phenotypes of spt7 and spt15 mutants, including effects of spt7 mutations on the transcription start site of certain genes, suggests that SPT7 plays an important role in transcription initiation in vivo.

Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C.

A set of GAL2+ yeast strains that are isogenic to strain S288C have been constructed. They contain non-reverting mutations in genes commonly used for selection for recombinant plasmids. Strains from this collection are being used for the European Union Yeast Genome Sequencing Programme. Representative strains from this collection have been deposited with the ATCC.

SPT10 and SPT21 are required for transcription of particular histone genes in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome contains four loci that encode histone proteins. Two of these loci, HTA1-HTB1 and HTA2-HTB2, each encode histones H2A and H2B. The other two loci, HHT1-HHF1 and HHT2-HHF2, each encode histones H3 and H4. Because of their redundancy, deletion of any one histone locus does not cause lethality. Previous experiments demonstrated that mutations at one histone locus, HTA1-HTB1, do cause lethality when in conjunction with mutations in the SPT10 gene. SPT10 has been shown to be required for normal levels of transcription of several genes in S. cerevisiae. Motivated by this double-mutant lethality, we have now investigated the interactions of mutations in SPT10 and in a functionally related gene, SPT21, with mutations at each of the four histone loci. These experiments have demonstrated that both SPT10 and SPT21 are required for transcription at two particular histone loci, HTA2-HTB2 and HHF2-HHT2, but not at the other two histone loci. These results suggest that under some conditions, S. cerevisiae may control the level of histone proteins by differential expression of its histone genes.

The Saccharomyces cerevisiae SPT8 gene encodes a very acidic protein that is functionally related to SPT3 and TATA-binding protein.

Mutations in the Saccharomyces cerevisiae SPT8 gene were previously isolated as suppressors of retrotransposon insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Mutations in SPT8 confer phenotypes similar to those caused by particular mutations in SPT15, which encodes the TATA-binding protein (TBP). These phenotypes are also similar to those caused by mutations in the SPT3 gene, which encodes a protein that directly interacts with TBP. We have now cloned and sequenced the SPT8 gene and have shown that it encodes a predicted protein of 602 amino acids with an extremely acidic amino terminus. In addition, the predicted SPT8 amino acid sequence contains one copy of a sequence motif found in multiple copies in a number of other eukaryotic proteins, including the beta subunit of heterotrimeric G proteins. To investigate further the relationship between SPT8, SPT3 and TBP, we have analyzed the effect of an spt8 null mutation in combination with different spt3 and spt15 mutations. This genetic analysis has shown that an spt8 deletion mutation is suppressed by particular spt3 alleles. Taken together with previous results, these data suggest that the SPT8 protein is required, directly or indirectly, for TBP function at particular promoters and that the role of SPT8 may be to promote a functional interaction between SPT3 and TBP.

The products of the SPT10 and SPT21 genes of Saccharomyces cerevisiae increase the amplitude of transcriptional regulation at a large number of unlinked loci.

The 3' long terminal repeat (LTR) of yeast transposon Ty1 is not normally used as a promoter, although it contains sequences identical to those found in the 5' LTR, which does act as a promoter. We have isolated mutations that fall into two genes, SPT10 and SPT21, that allow the 3' LTRs of Ty1 elements inserted at various positions in the genome of Saccharomyces cerevisiae to act as promoters. We find that mutations in these two genes alter transcriptional regulation of Ty1 LTRs and also of certain non-Ty1-related promoters in two ways: (i) they allow the low-level expression of several genes under repressing conditions, and (ii) they allow transcription from the 5' LTR of Ty1 elements in the absence of a normally required activator, SPT3. Furthermore, the fully induced levels of transcription of several genes are reduced in these spt mutants. Hence, the products of these two genes increase the amplitude of transcriptional regulation of a wide variety of unlinked loci.

SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcription initiation in vivo.

Mutations in the S. cerevisiae SPT15 gene were isolated as suppressors of insertion mutations that alter the transcription of adjacent genes. Molecular and genetic analysis of the cloned SPT15 gene has shown that it is the same as the gene that encodes the TATA binding factor TFIID. Analysis of spt15 mutants has demonstrated that alterations in TFIID can change transcription initiation in vivo. In addition, we demonstrate that TFIID is essential for growth and that spt15 mutations are pleiotropic, as spt15 mutants grow slowly and have defects in both mating and sporulation. Therefore, TFIID is an essential transcription factor in vivo, likely to be required for normal expression of a large number of genes.

Cyclic AMP-dependent protein kinase phosphorylates and inactivates the yeast transcriptional activator ADR1.

It has been proposed in several eukaryotic systems that the regulation of gene transcription involves phosphorylation of specific transcription factors. We report here that the yeast transcriptional activator ADR1 is phosphorylated in vitro by cyclic AMP-dependent protein kinase and that mutations which enhance the ability of ADR1 to activate ADH2 expression decrease ADR1 phosphorylation. We also show that increased kinase activity in vivo inhibits ADH2 expression in an ADR1 allele-specific manner. Our data suggest that glucose repression of ADH2 is in part mediated through a cAMP-dependent phosphorylation-inactivation of the ADR1 regulatory protein.

Three genes are required for trans-activation of Ty transcription in yeast.

Mutations in the SPT3 gene were isolated as one class of suppressors of Ty and solo delta insertion mutations in Saccharomyces cerevisiae. Previous work has shown that null mutations in SPT3 abolish the normal Ty delta-delta transcript; instead, a transcript that initiates 800 bases farther downstream is made, suggesting that SPT3 is required for transcription initiation in delta sequences. We have selected for new spt mutations and have screened for those with the unique suppression pattern of spt3 mutations with respect to two insertion mutations. Our selection and screen has identified two additional genes, SPT7 and SPT8, that are also required for transcription initiation in delta sequences. We show that mutations in SPT7 or SPT8 result in the same alteration of Ty transcription as do mutations in SPT3. In addition, mutations in all three genes cause a sporulation defect. By assay of a Ty-lacZ fusion we have shown that spt3, spt7 and spt8 mutations reduce transcription from a delta sequence by 10-25-fold. Finally, we show that SPT3 mRNA levels are unaffected in either spt7 or spt8 mutants, suggesting that these two genes do not regulate transcription of SPT3.