Allograft inflammatory factory-1. A cytokine-responsive macrophage molecule expressed in transplanted human hearts.

Allograft inflammatory factor-1 (AIF-1), a cytokine-responsive macrophage molecule, was originally identified and cloned from rat cardiac allografts with chronic cardiac rejection. We performed the present study to determine whether AIF-1 was also involved in the inflammatory response associated with human cardiac transplant rejection. AIF-1 gene transcripts were identified by the reverse-transcriptase polymerase chain reaction in endomyocardial biopsy specimens from human heart transplants and in macrophage cell lines. In the 441-base pair coding region of the human and rat cDNAs, the nucleotide sequences were 86% identical and the deduced amino acid sequences were 90% identical. Consistent with our studies in the rat. AIF-1 was selectively expressed in human macrophage-like cell lines, and immunostaining in human heart allografts localized the AIF-1 gene product to a subset of CD68+ macrophages in the interstitial and perivascular spaces. The parallels between rat and human AIF-1 expression suggest that AIF-1 may have a common effect on the function of activated macrophages in cardiac allografts.

Cloning and characterization of allograft inflammatory factor-1: a novel macrophage factor identified in rat cardiac allografts with chronic rejection.

The development of arteriosclerotic lesions in the Lewis to F344 rat model of chronic cardiac rejection is characterized by macrophage adhesion to the vessel lumen and macrophage infiltration in the neointima prior to smooth muscle cell accumulation. We report the cloning and characterization of allograft inflammatory factor-1 (AIF-1), a novel cDNA that is expressed early and persistently in chronically rejecting cardiac allografts but is absent in cardiac syngrafts and host hearts. The full-length cDNA codes for a hydrophilic polypeptide of 17 kD that contains a 12-amino acid region similar to an EF-hand (calcium-binding) domain. In cardiac allografts AIF-1 transcripts and protein localized to infiltrating mononuclear cells. Analysis of isolated cell populations confirmed that AIF-1 was selectively expressed in macrophages and neutrophils and demonstrated that AIF-1 transcripts could be upregulated by sixfold after stimulation with the T cell-derived cytokine IFN-gamma. Treatment with a diet deficient in essential fatty acids (which attenuates arteriosclerosis) or CTLA-4 Ig (which blocks lymphocyte activation) significantly decreased AIF-1 transcript levels. Upregulation of AIF-1 in the setting of T cell activation suggests that it may play a role in macrophage activation and function.

Identification and upregulation of galactose/N-acetylgalactosamine macrophage lectin in rat cardiac allografts with arteriosclerosis.

Using differential mRNA display to uncover potential mediators associated with chronic rejection, we identified a cDNA fragment induced in Lewis to F344 rat cardiac allografts with arteriosclerosis but not Lewis syngrafts. The full-length cDNA (1.4 kb) isolated from a rat cardiac allograft cDNA library was 99% identical to galactose/N-acetylgalactosamine (Gal/GalNAc) macrophage lectin, a cell-surface receptor. This cDNA hybridized in Northern analysis with total RNA from eight cardiac allografts but not with host hearts, syngrafts, or other organs. There was a significant allograft-specific increase in transcript levels measured by reverse transcriptase PCR at days 7, 14, 28, and 75 in comparison with paired F344 host hearts (subject to same circulation but histologically normal), day-0 hearts, and syngrafts (P < 0.008, n = 4 at each time). Transcript levels in cardiac allografts were higher than those in paired host spleens (a major source of inflammatory cells) (P < 0.0001), indicating the localized nature of Gal/GalNAc lectin induction. By in situ hybridization and immunostaining, Gal/GalNAc lectin expression localized to a subset of inflammatory cells in cardiac allografts. These findings link Gal/GalNAc macrophage lectin to the chronic rejection process, as a possible mediator of macrophage infiltration.

Chronic cardiac rejection: identification of five upregulated genes in transplanted hearts by differential mRNA display.

Transplant arteriosclerosis, the major manifestation of chronic rejection, develops after allogeneic (Lewis to F344) but not syngeneic (Lewis to Lewis) rat cardiac transplantation. To identify transcriptionally regulated mediators associated with chronic cardiac rejection, we adapted the differential mRNA display technique for in vivo transplant specimens. Gene transcript patterns in four allogeneic hearts showing early signs of chronic rejection were compared with those in two syngeneic hearts exposed to the same surgical procedure but histologically normal. Twelve differentially expressed cDNA bands were identified. We improved the probability of isolating one or more allograft-specific cDNAs from a single display band by first using recovered and reamplified PCR products as probes in RNA blot analysis. cDNA fragments cloned from individual bands were then used in a second RNA blot analysis, which allowed for the correlation of specific mRNA transcripts with cDNA clones. Five cDNA clones produced time-dependent, allograft-specific hybridization. Sequence analysis demonstrated that two of these cDNAs corresponded to unknown genes, whereas the other three represented known genes not previously associated with chronic rejection. The latter group included the macrophage lectin specific for galactose/N-acetylgalactosamine (a cell-surface receptor), the nuclear P1 gene (a homologue of a yeast replication protein), and a ubiquitin-like gene. Our application of the differential display technique allowed the direct identification of potential mediators under in vivo conditions that preserve the environment of the disease process--including infiltrating cell populations critical to the inflammatory response.

A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple snRNP-specific proteins.

One of the consequences of the heat shock response is a shutdown of pre-mRNA splicing, a phenomenon that can be reproduced in extracts prepared from heat-shocked cells. The block in splicing occurs before the covalent modifications that generate spliced mRNA at the level of spliceosome formation. We have used extracts prepared from heat-shocked cells as a complementation system to characterize and partially purify a protein factor that is inactivated during the in vivo heat shock. The activity functions in the formation of the active spliceosome by assembling U4/U6 and U5 snRNPs into a triple snRNP particle. The factor appears to be different from previously isolated splicing factors and is functionally equivalent to several polypeptides that are specifically associated with the purified triple snRNP but not with individual U4/U6 or U5 snRNPs. Our data confirm the hypothesis that U4/U6 and U5 snRNPs enter the spliceosome as a triple snRNP complex and show for the first time a function of specific snRNP-associated polypeptides in the mammalian splicing pathway.

Identification of cis-acting intron and exon regions in influenza virus NS1 mRNA that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes.

In in vitro splicing reactions, influenza virus NS1 mRNA was not detectably spliced, but nonetheless very efficiently formed ATP-dependent 55S complexes containing the U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) (C. H. Agris, M. E. Nemeroff, and R. M. Krug, Mol. Cell. Biol. 9:259-267, 1989). We demonstrate that the block in splicing was caused by two regions in NS1 mRNA: (i) a large intron region (not including the branchpoint sequence) and (ii) an 85-nucleotide 3' exon region near the 3' end of the exon. After removal of both of these regions, the 5' and 3' splice sites and branchpoint of NS1 mRNA functioned efficiently in splicing, indicating that they were not defective. The two inhibitory regions shared one property: splicing inhibition was independent of the identity of the nucleotide sequence in either region. In other respects, however, the two inhibitory regions differed. The inhibitory activity of the intron region was proportional to its length, indicating that the inhibition was probably due to size only. In contrast, the 3' exon, which was of small size, was a context element; i.e., it functioned only when it was located at a specific position in the 3' exon of NS1 mRNA. To determine how these intron and exon regions inhibited splicing, we compared the types of splicing complexes formed by intact NS1 mRNA with those formed by spliceable NS1 mRNA lacking the intron and exon regions. Splicing complexes were formed by using purified splicing factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Three protein factors (SF1, SF3 and U2AF) function in pre-splicing complex formation in addition to snRNPs.

The splicing of nuclear messenger RNA precursors can be reproduced in vitro with fractions obtained after chromatography of HeLa cell nuclear extracts. Here we report the chromatographic separation of three protein factors: SF1, SF3 and U2AF. All factors function early in the splicing reaction, in the assembly of a pre-splicing complex. Likewise, all factors are essential for the production of spliced RNA. In addition to their distinct chromatographic properties, the splicing factors can be distinguished by their sensitivities to heat and N-ethylmaleimide. All activities can be detected in a cytoplasmic A-100 fraction from HeLa cells. The fact that SF1, SF3 and U2AF are essential factors in pre-splicing complex formation raises the possibility that SF1 and/or SF3 participate in the interaction of U2 snRNP with the branch point in addition to U2AF.

Splicing factor SF4 is dispensable for the assembly of a functional splicing complex and participates in the subsequent steps of the splicing reaction.

The splicing of nuclear messenger RNA precursors (pre-mRNA) can be reconstituted in vitro with factors partially purified from HeLa cell nuclear extracts. Splicing complexes are assembled in the presence of the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6 and the protein factors SF1, SF2, SF3 and U2AF. However, the complexes thus formed are inactive, i.e. they only contain unprocessed pre-mRNA. The intermediates and products of the splicing reaction are generated after addition of SF4. This splicing factor is a heat-labile protein which requires sulfhydryl groups for its activity. SF4 appears to participate, directly or indirectly, in the conversion of a functional but inactive splicing complex to the active spliceosome.