Induction of members of the IL-8/NAP-1 gene family in human T lymphocytes is suppressed by cyclosporin A.

Human members of a family of structurally related cytokines, which play a role as effectors of inflammation, were analyzed for their expression and regulation in T lymphocytes. Members of this gene family include Platelet Basic Protein (PBP); Platelet Factor 4 (PF-4); IL-8/NAP-1; IP-10, a gamma interferon induced protein; GRO; pAT 464 and pAT 744. In resting T lymphocytes the RNAs of the individual genes could not be detected, but all genes were induced upon stimulation with PHA or with PHA/PMA. The induction of five genes was blocked by the immunosuppresive drug cyclosporin A (CSA), which appears to affect initial events in T cell activation. This expression in T lymphocytes, especially the sensitivity to CSA, indicates a common immunmodulatory role of these structural related proteins.     

A family of small inducible proteins secreted by leukocytes are members of a new superfamily that includes leukocyte and fibroblast-derived inflammatory agents, growth factors, and indicators of various activation processes

We have isolated and characterized four cDNA clones that encode mRNA expressed more abundantly in Con A-activated mouse helper T cells than by resting T cells. One mRNA encoded a approximately 14-kDa protein with a hydrophobic N-terminal sequence and was abundantly expressed by the Th 2 subset of Th cells, but was not expressed by Th 1 cells. The remaining three mRNA encoded related approximately 8-kDa secreted proteins that are part of a family of small, secreted, and inducible mouse and human proteins. This family of proteins is itself distantly related to another family of growth and inflammatory factors that are associated with various lymphoid and fibroblast activation phenomena. One of the small, inducible, secreted proteins has a predicted mature N terminus identical to that of the previously described macrophage inflammatory protein.     

A novel polypeptide secreted by activated human T lymphocytes

We have identified two cDNA clones, I-309 and G-26, which define genes expressed abundantly in activated human PBMC, but at low or undetectable levels in resting PBMC. Based upon nucleotide sequence analysis, both clones are predicted to encode small, structurally related polypeptides, each containing a hydrophobic leader sequence characteristic of secreted proteins and a motif of four conserved cysteine residues. Further, I-309 and G-26 are structurally related to a growing family of genes that apparently encode small polypeptides whose secretion is induced upon cell activation. I-309 represents a previously undescribed human gene. We have generated an anti-peptide antiserum to the I-309 gene product which recognizes proteins in culture supernatants of an activated T cell clone and of COS cells transfected with the I-309 cDNA, supporting the idea that I-309 encodes a secreted protein. Because I-309 encodes a small protein secreted by activated T cells that displays structural features similar to other cytokines, we believe that it defines a novel cytokine with as yet unknown function.     

Subpopulations of lymphocytes to produce various lymphokines. I. Function of subpopulations separated by velocity sedimentation.

Identification of a subpopulation of lymphocytes producing lymphokines was attempted by fractionating the lymph node cells from guinea pigs immune to DNP-BSA by velocity sedimentation at 1 x G. Each of six fractions obtained by this procedure was cultured with or without the presence of antigen, and the culture supernatants that were separated 24 hr later were assayed for various lymphokine activities. Most of the lymphokines, including migration inhibition factor, chemotactic factor for neutrophils, mitogenic factor, and lymphotoxin were generated by the first two fractions of lymphocytes, which represented the largest, most rapidly sedimenting cells. Although th procedure of cell separation does not depend on cell surface properties, the larger cells contained more cells with T cell surface markers and the smalller contained more cells with B cell surface markers. Proliferative response of those lymphocytes measured by 3H-thymidine uptake, however, has shown that the largest two subpopulations responded poorly either to specific antigens or to mitogens (PHA and LPS), and rather that the medium size cells responded most strongly to the both stimulants. These results indicated that the production of some lymphokines confined to certain subpopulations of lymphocytes which are large in size. Further, these cells are readily separable from the medium sized cells that respond strongly to antigenic and mitogenic stimuli with mitogenic responses.     

A macrophage LPS-inducible early gene encodes the murine homologue of IP-10

Recently, we have isolated and characterized a set of cDNA clones which encode lipopolysaccharide-inducible proteins in murine peritoneal macrophages. Here, we report the sequence and identification of one of these cDNAs previously termed C7. Nucleotide sequence analysis revealed an open reading frame encoding a predicted polypeptide composed of 98 amino acids, which contained a 21 amino acid residue signal peptide, indicating approximately 9 kDa of mature protein. The deduced protein sequence showed homology (67% identity, 77% considering conservative amino acid changes) with the human INF gamma-inducible gene IP-10, a member of the recently described superfamily of chemotactic and mitogenic proteins which includes platelet factor 4, monocyte-derived neutrophil chemotactic factor (NAF, NAP-1, IL-8), and MGSA/gro/KC. Thus C7 would appear to represent the murine homologue of the human IP-10 gene or a very closely related gene.     

Recombinant expression, biochemical characterization, and biological activities of the human MGSA/gro protein

Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterization shows that the recombinant protein is identical with the natural protein and is devoid of posttranslational glycosylation, sulfation, and phosphorylation. The two C-terminal amino acids are proteolytically removed from the mature recombinant MGSA, indicating a length of 71 instead of the predicted 73 amino acids. The recombinant MGSA is mitogenically active on the Hs294T melanoma cells. The purified MGSA competes with interleukin 8 for binding to neutrophil receptors and exhibits neutrophil chemotactic activity equivalent to that of interleukin 8.     

Diversity of Arabidopsis Genes Encoding Precursors for Phytosulfokine, a Peptide Growth Factor

Phytosulfokine-alpha (PSK-alpha), a unique plant peptide growth factor, was originally isolated from conditioned medium of asparagus (Asparagus officinalis) mesophyll cell cultures. PSK-alpha has several biological activities including promoting plant cell proliferation. Four genes that encode precursors of PSK-alpha have been identified from Arabidopsis. Analysis of cDNAs for two of these, AtPSK2 and AtPSK3, shows that both of these genes consist of two exons and one intron. The predicted precursors have N-terminal signal peptides and only a single PSK-alpha sequence located close to their carboxyl termini. Both precursors contain dibasic processing sites flanking PSK, analogous to animal and yeast prohormones. Although the PSK domain including the sequence of PSK-alpha and three amino acids preceding it are perfectly conserved, the precursors bear very limited similarity among Arabidopsis and rice (Oryza sativa), suggesting a new level of diversity among polypeptides that are processed into the same signaling molecule in plants, a scenario not found in animals and yeast. Unnatural [serine-4]PSK-beta was found to be secreted by transgenic Arabidopsis cells expressing a mutant of either AtPSK2 or AtPSK3 cDNAs, suggesting that both AtPSK2 and AtPSK3 encode PSK-alpha precursors. AtPSK2 and AtPSK3 were expressed demonstrably not only in cultured cells but also in intact plants, suggesting that PSK-alpha may be essential for plant cell proliferation in vivo as well as in vitro. Overexpression of either precursor gene allowed the transgenic calli to grow twice as large as the controls. However, the transgenic cells expressing either antisense cDNA did not dramatically decrease mitogenic activity, suggesting that these two genes may act redundantly.     

Platelet-derived growth factor-inducible gene JE is a member of a family of small inducible genes related to platelet factor 4

The platelet-derived growth factor-inducible gene JE was found to encode a 148-residue basic (pI = 10.4) secretory protein which shows striking similarity to the gene products of a family of small inducible genes (SIG), LD78, TCA3, IP10, 3-10C, 9E3/pCEF4, and gro/MGSA, and to several of the proteins secreted from platelet alpha-granules. Members of the SIG family have spatially conserved cysteine residues that vary in distance by only one amino acid residue as well as conserved proline residues at analogous sites. Hydrophilicity plots show alternating hydrophobic and hydrophilic domains which are similar for all members of the SIG family except IP10 and platelet factor 4, which show similarities to each other. The genomic organization of SIG family members is similar in the location of the splice junctions and the number of introns and exons, suggesting that they were derived from a common ancestor. The collective evidence suggests that a family of inducible cytokines, which are mitogenic or chemotactic, may act as intercellular coordinators of diverse responses designed to combat infection and promote the healing and regeneration of injured tissue.     

A novel immediate-early response gene of endothelium is induced by cytokines and encodes a secreted protein.

We have previously described the cloning of a group of novel cellular immediate-early response genes whose expression in human umbilical vein endothelial cells is induced by tumor necrosis factor alpha in the presence of cycloheximide. These genes are likely to participate in mediating the response of the vascular endothelium to proinflammatory cytokines. In this study, we further characterized one of these novel gene products named B61. Sequence analysis of cDNA clones encoding B61 revealed that its protein product has no significant homology to previously described proteins. Southern analysis suggested that B61 is an evolutionarily conserved single-copy gene. B61 is primarily a hydrophilic molecule but contains both a hydrophobic N-terminal and a hydrophobic C-terminal region. The N-terminal region is typical of a signal peptide, which is consistent with the secreted nature of the protein. The mature form of the predicted protein consists of 187 amino acid residues and has a molecular weight of 22,000. Immunoprecipitation of metabolically labeled human umbilical vein endothelial cell preparations revealed that B61 is a 25-kilodalton secreted protein which is markedly induced by tumor necrosis factor.     

Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10

Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media. Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media. Immunoblot analysis of the affinity-purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides. Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF-related mitogen secreted by human vascular endothelial cells. The protein has a 45% overall homology to the translation product of the v-src-induced CEF-10 mRNA from chick embryo fibroblasts. We have termed this new mitogen connective tissue growth factor.     

Yersinia enterocolitica activates a T helper type 1-like T cell subset in reactive arthritis.

The profile of lymphokines secreted by 14 T cell clones and 24 T cell lines reactive with Yersinia Ag isolated from the synovial fluid cells of two HLA-B27+ patients with Yersinia-triggered reactive arthritis was characterized. In response to Ag-specific or -nonspecific stimulation, all of the Yersinia-reactive T cell clones and lines had a pattern of lymphokine secretion resembling that of murine (Th1) cells. A total of 50% of T cell lines and clones randomly isolated from a reactive arthritis patient, without prior in vitro stimulation with Yersinia Ag, also exhibited a Th1-like profile of cytokine secretion upon nonspecific activation. This indicates that the selective expansion of this subset of T cells had already occurred in vivo. The possibility that the predominance of Th1-like T cells was an artefact generated by the T cell cloning procedure was excluded; 50% of the randomly isolated T cell clones and lines produced IL-4, IL-5, or both cytokines upon nonspecific activation. These results indicate that Yersinia Ag selectively activate a Th1-like subset of T cells in patients with Yersinia-triggered reactive arthritis. Accumulation of such cells in the synovial tissue of patients with reactive arthritis may play a key role in the pathogenesis of this disease.     

Mechanisms in the in vivo release of lymphokines. 1. Comparative kinetics in the release of six lymphokines in inbred strains of mice

Lymphokines were detected in the sera of 16 strains of inbred mice sensitized intravenously with cell walls of Mycobacterium bovis strain BCG and challenged subsequently intravenously with old tuberculin (OT). Variations occurred between the strains in the types and quantities of six lymphokines studied, namely, chemotactic factor (CF), type II interferon (IF II), lymphotoxin (LT), migration inhibitory factor (MIF), mitogenic factor (MF), and skin reactive factor (SRF). The times for maximum release of the lymphokines in the different strains were similar for MIF, IF II, SRF, and MF, but differed for CF and LT. The degree of activity of MIF and IF II generally paralleled one another in the different strains but such parallelism did not occur for the other four lymphokines. Each of the 16 strains was a high responder for at least one of the lymphokines, indicating that in sensitized mice the release or presence in the circulation of lymphokines in response to a specific antigen is a selective process. Thus, each strain may have an individual combination of lymphokines, interactions of which may determine the types of pathway utilized in an immunological response.     

Characterization of T cell clones from an athymic mouse.

Although Thy-1+ lymphocytes have been observed in lymphoid tissues of athymic mice, attempts to analyze these cells on the clonal level have previously yielded only populations of CD4-CD8+ cytolytic T cells. Furthermore, studies of responses of these cells to various mitogenic stimuli have demonstrated significant defects in the ability of these cells to proliferate in culture. We report here on the cloning and maintenance in long term culture of T cells from an athymic mouse stimulated in vitro with allogeneic spleen cells. Of 10 Thy-1+ clones, 7 CD4+CD8- and 3 CD4-CD8+ Ag-specific cells were obtained. Among the CD4+ T cells, we observed a variety of specificities, including an autoreactive I-Aq specific clone, a minor lymphocyte stimulating determinant (Mls)-reactive clone, and five allo-I-Ad-specific CD4+ clones; a class II-specific CD4-CD8+ clone was also obtained. In addition, we observed two Thy-1-CD3+ clones (one of which is also CD4+ and expresses V beta 8) which are constitutively responsive to the lymphokines IL-2 and IL-4. Of 11 clones tested, 7 produce IL-2 and/or IL-4 lymphokines after stimulation through the TCR, whereas 4 do not, requiring exogenous lymphokines for optimal responses to Ag. Of 10 clones tested for IL-2R expression, 3 had notably low levels, correlating with low proliferative responses to IL-2. The results reveal the spectrum of T cells available to a mouse which is congenitally athymic and describe the heterogeneity of immune defects expressed in such cells at the clonal level.     

Identification and characterization of host factors interacting with Bombyx mori nucleopolyhedrovirus ORF8

The orf8 gene (Bm8) in Bombyx mori nucleopolyhedrovirus (BmNPV) is one of 17 genes unique to group I NPVs and is expressed as an early gene. We have reported that Bm8 may play an important role during viral infection and that Bm8 protein co-localized with IE1 to specific nuclear foci throughout infection. It was also demonstrated that both IE1 and BmNPV hr facilitate this localization of Bm8. To investigate further, host proteins interacting with Bm8 were screened using a yeast two-hybrid system. We identified 6 host clones as Bm8-interacting partners from three cDNA libraries derived from BmN cells or B. mori larvae. Further assays showed that the N-terminal region of Bm8 is important for the interaction with most host clones and that two of the clones can associate with IE1. Cloning and sequencing of full-length cDNAs revealed that most of the clones potentially encode either membrane-bound proteins or secreted proteins. Quantitative RT-PCR analysis revealed that some of these host genes were slightly induced during the early stage of infection in BmN cells, and that the expression of all genes was markedly reduced during the late stage of infection. Generation of mutant BmNPVs over-expressing these host genes also identified a gene that potentially functions as a negative factor during BmNPV infection. These features of Bm8-interacting host proteins strongly support that Bm8 is a multifunctional protein involved in multiple signaling pathways in host cells.     

Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones

The effect of glucocorticoids on lymphokine production by T lymphocytes was examined by using long-term alloreactive T cell clones that secreted one or more of the lymphokines interleukin 2 (IL 2), interferon-gamma, macrophage-activating factor (MAF), and colony-stimulating factor when stimulated by an antigen or a mitogen. Production of all of these four lymphokines was inhibited when glucocorticoids were added at physiologic concentrations (10(-8) to 10(-6) M) to clones stimulated with concanavalin A (Con A). Clones were heterogeneous with respect to their sensitivity to glucocorticoid inhibition of MAF production; cytolytic clones were generally more resistant than noncytolytic clones. The glucocorticoid dexamethasone (Dex) and an IL 2-containing supernatant exerted opposing effects on clonal MAF production. Kinetics experiments showed that Dex inhibited MAF production by reducing the rate of secretion without causing a compensatory increase in the duration of secretion, whereas the IL 2 source increased the rate and the total amount of MAF secretion. Dex abrogated the effect of IL 2. Inhibition by Dex was apparent from the earliest time of detectable MAF production (about 4 hr after stimulation) and increased with longer exposure until production ceased (12 to 24 hr). Pre-exposure and removal of Dex before Con A stimulation also inhibited MAF release. Effects of Dex on lymphokine secretion by clones could be dissociated from effects on their growth in response to stimulator cells and IL 2. Factor production by the 16 clones tested was inhibited to some degree. Proliferation, however, by two of these clones (both cytolytic) was unaffected by Dex, whereas proliferation of two noncytolytic clones was strongly inhibited even in the presence of a saturating dose of IL 2.