Anti-nuclear antibody production and immune-complex glomerulonephritis in BALB/c mice treated with pristane.

The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.

Amelioration of lupus-like autoimmune disease in NZB/WF1 mice after treatment with a blocking monoclonal antibody specific for complement component C5.

New Zealand black x New Zealand white (NZB/W) F1 mice spontaneously develop an autoimmune syndrome with notable similarities to human systemic lupus erythematosus. Female NZB/WF1 mice produce high titers of antinuclear antibodies and invariably succumb to severe glomerulonephritis by 12 months of age. Although the development of the immune-complex nephritis is accompanied by abundant local and systemic complement activation, the role of proinflammatory complement components in disease progression has not been established. In this study we have examined the contribution of activated terminal complement proteins to the pathogenesis of the lupus-like autoimmune disease. Female NZB/W F1 mice were treated with a monoclonal antibody (mAb) specific for the C5 component of complement that blocks the cleavage of C5 and thus prevents the generation of the potent proinflammatory factors C5a and C5b-9. Continuous therapy with anti-C5 mAb for 6 months resulted in significant amelioration of the course of glomerulonephritis and in markedly increased survival. These findings demonstrate an important role for the terminal complement cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated C5 inhibition may be a useful approach to the therapy of immune-complex glomerulonephritis in humans.

Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin⋅FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.

The tobacco wounding-activated mitogen-activated protein kinase is encoded by SIPK

It has been demonstrated that both salicylic acid and fungal elicitors activate a 48-kDa mitogen-activated protein kinase termed salicylic acid-induced protein kinase (SIPK) in tobacco suspension cells. Here, we show that infiltration of these agents into tobacco leaves also activates SIPK. Of particular interest, infiltration of water alone activated a kinase of the same size, possibly because of wounding and/or osmotic stresses. The kinetics of kinase activation, however, differ for these different treatments. Various mechanical stresses, including cutting and wounding by abrasion, also activated a 48-kDa kinase. By using an immune-complex kinase assay with antibodies specific for SIPK or wounding-induced protein kinase, we demonstrate that this wounding-activated 48-kDa kinase is SIPK, rather than wounding-induced protein kinase, as reported [Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. & Ohashi, Y. (1995) Science 270, 1988–1992]. Activation of SIPK after wounding was associated with tyrosine phosphorylation but not with increases in SIPK mRNA or protein levels. Thus, the same mitogen-activated protein kinase, SIPK, appears to facilitate signaling for two distinct pathways that lead to disease resistance responses and wounding responses.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Wortmannin-Sensitive Phosphorylation, Translocation, and Activation of PLCγ1, but Not PLCγ2, in Antigen-stimulated RBL-2H3 Mast Cells

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcεRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcεRI cross-linking activates both PLCγ1 and PLCγ2. Activation is accompanied by the increased phosphorylation of both PLCγ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCγ isoforms have distinct subcellular localizations. PLCγ1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCγ1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCγ2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcεRI cross-linking. The activation of PLCγ1, but not of PLCγ2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCγ1 with little inhibition of PLCγ2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCγ1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCγ1. Although less abundant than PLCγ2, activated PLCγ1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.

Cyclin D3-associated Kinase Activity Is Regulated by p27kip1 in BALB/c 3T3 Cells

We report that cyclin D3/cdk4 kinase activity is regulated by p27kip1 in BALB/c 3T3 cells. The association of p27kip1 was found to result in inhibition of cyclin D3 activity as measured by immune complex kinase assays utilizing cyclin D3-specific antibodies. The ternary p27kip1/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27kip1-specific antibodies. The association of p27kip1 with cyclin D3 was highest in quiescent cells and declined upon mitogenic stimulation, concomitantly with declines in the total level of p27kip1 protein. The decline in this association could be elicited by PDGF treatment alone; this was not sufficient, however, for activation of cyclin D3 activity, which also required the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27kip1 kinase activity was present throughout the cell cycle. Since we found that the p27kip1 activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active ternary complex containing p27kip1 and the active cyclin D3 lacking p27kip1 by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitro and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27kip1/cyclin D3/cdk4 complexes exhibited a different specificity than the active binary cyclin D3/cdk4 complexes, suggesting that p27kip1 has the capacity to both inhibit cyclin D/cdk4 activity as well as to modulate cyclin D3/cdk4 activity by altering its substrate preference.

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Δ cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Δ cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.

Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation.

Urea (200-400 milliosmolar) activates transcription, translation of, and trans-activation by the immediate-early gene transcription factor Egr-1 in a renal epithelial cell-specific fashion. The effect at the transcriptional level has been attributed to multiple serum response elements and their adjacent Ets motifs located within the Egr-1 promoter. Elk-1, a principal ternary complex factor and Ets domain-containing protein, is a substrate of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In the renal medullary mIMCD3 cell line, urea (200-400 milliosmolar) activated both ERK1 and ERK2 as determined by in-gel kinase assay and immune-complex kinase assay of epitope-tagged] ERK1 and ERK2. Importantly, urea did not affect abundance of either ERK. Urea-inducible Egr-1 transcription was a consequence of ERK activation because the ERK-specific inhibitor, PD98059, abrogated transcription from the murine Egr-1 promoter in a luciferase reported gene assay. In addition, activators of protein kinase A, including forskolin and 8-Br-cAMP, which are known to inhibit ERK-mediated events, also inhibited urea-inducible Egr-1 transcription. Furthermore, urea-inducible activation of the physiological ERK substrate and transcription factor, Elk-1, was demonstrated through transient cotransfection of a chimeric Elk-1/GAL4 expression plasmid and a GAL4-driven luciferase reporter plasmid. Taken together, these data indicate that, in mIMCD3 cells, urea activates ERKs and the ERK substrate, Elk-1, and that ERK inhibition abrogates urea-inducible Egr-1 transcription. These data are consistent with a model of urea-inducible renal medullary gene expression wherein sequential activation of ERKs and Elk-1 results in increased transcription of Egr-1 through serum response element/Ets motifs.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter.

Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA–liposome complexes

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA–liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.

Diversification, expression, and γδ T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial γδ T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative α-helical regions of the α1α2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their α1α2 domains, they were recognized by diverse human intestinal epithelial γδ T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of γδ T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.

Mycobacterium avium complex augments macrophage HIV-1 production and increases CCR5 expression

Infection with HIV-1 results in pronounced immune suppression and susceptibility to opportunistic infections (OI). Reciprocally, OI augment HIV-1 replication. As we have shown for Mycobacterium avium complex (MAC) and Pneumocystis carinii, macrophages infected with opportunistic pathogens and within lymphoid tissues containing OI, exhibit striking levels of viral replication. To explore potential underlying mechanisms for increased HIV-1 replication associated with coinfection, blood monocytes were exposed to MAC antigens (MAg) or viable MAC and their levels of tumor necrosis factor α (TNFα) and HIV-1 coreceptors monitored. MAC enhanced TNFα production in vitro, consistent with its expression in coinfected lymph nodes. Using a polyclonal antibody to the CCR5 coreceptor that mediates viral entry of macrophage tropic HIV-1, a subset of unstimulated monocytes was shown to be CCR5-positive by fluorescence-activated cell sorter analysis. After stimulation with MAg or infection with MAC, CCR5 expression was increased at both the mRNA level and on the cell surface. Up-regulation of CCR5 by MAC was not paralleled by an increase in the T cell tropic coreceptor, CXCR4. Increases in NF-κB, TNFα, and CCR5 were consistent with the enhanced production of HIV-1 in MAg-treated adherent macrophage cultures as measured by HIV-1 p24 levels. Increased CCR5 was also detected in coinfected lymph nodes as compared with tissues with only HIV-1. The increased production of TNFα, together with elevated expression of CCR5, provide potential mechanisms for enhanced infection and replication of HIV-1 by macrophages in OI-infected cells and tissues. Consequently, treating OI may inhibit not only the OI-induced pathology, but also limit the viral burden.

Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis

The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene B4 biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.