Pleiotropic contributions of phospholipase C-gamma1 (PLC-gamma1) to T-cell antigen receptor-mediated signaling: reconstitution studies of a PLC-gamma1-deficient Jurkat T-cell line.

Phospholipase C-gamma1 (PLC-gamma1) plays a crucial role in the coupling of T-cell antigen receptor (TCR) ligation to interleukin-2 (IL-2) gene expression in activated T lymphocytes. In this study, we have isolated and characterized two novel, PLC-gamma1-deficient sublines derived from the Jurkat T-leukemic cell line. The P98 subline displays a >90% reduction in PLC-gamma1 expression, while the J.gamma1 subline contains no detectable PLC-gamma1 protein. The lack of PLC-gamma1 expression in J.gamma1 cells caused profound defects in TCR-dependent Ca(2+) mobilization and NFAT activation. In contrast, both of these responses occurred at normal levels in PLC-gamma1-deficient P98 cells. Unexpectedly, the P98 cells displayed significant and selective defects in the activation of both the composite CD28 response element (RE/AP) and the full-length IL-2 promoter following costimulation with anti-TCR antibodies and phorbol ester. These transcriptional defects were reversed by transfection of P98 cells with a wild-type PLC-gamma1 expression vector but not by expression of mutated PLC-gamma1 constructs that lacked a functional, carboxyl-terminal SH2 [SH2(C)] domain or the major Tyr(783) phosphorylation site. On the other hand, the amino-terminal SH2 [SH2(N)] domain was not essential for reconstitution of RE/AP- or IL-2 promoter-dependent transcription but was required for the association of PLC-gamma1 with LAT, as well as the tyrosine phosphorylation of PLC-gamma1 itself, in activated P98 cells. These studies demonstrate that the PLC-gamma1 SH2(N) and SH2(C) domains play functionally distinct roles during TCR-mediated signaling and identify a non-Ca(2+)-related signaling function linked to the SH2(C) domain, which couples TCR plus phorbol ester-CD28 costimulation to the activation of the IL-2 promoter in T lymphocytes.

Polyomavirus middle T antigen as a probe for T cell antigen receptor-coupled signaling pathways.

Stimulation of the T cell antigen receptor (TCR) triggers a complex series of signaling events that culminate in T cell activation and proliferation. The complex structure of the TCR has hindered efforts to link specific signaling events induced by TCR cross-linkage to downstream activation responses, such as interleukin-2 (IL-2) gene transcription. Previous studies have shown that the polyomavirus-derived oncoprotein, middle T antigen (mT), transforms rodent fibroblasts by interacting with and activating several cytoplasmic signaling proteins (Src kinases, phospholipase C (PLC)-gamma1, Shc, and phosphoinositide 3-kinase (PI3-K) implicated in cell growth control. In this study, we demonstrate that expression of mT activates Jurkat T cells, as measured by increases in IL-2 promoter- and NFAT (nuclear factor of activated T cells)-dependent reporter gene transcription. The transcriptional response provoked by mT was blocked by the immunosuppressive drug FK506, a potent inhibitor of TCR-mediated IL-2 gene expression. Mutations that disrupted the binding of mT to Src kinases or PLC-gamma1 abrogated the ability of mT to deliver the signals needed for IL-2 promoter activation. In contrast, a mT mutant that failed to bind PI3-K induced a markedly elevated transcriptional response in Jurkat cells, whereas mutation of the Shc binding site in mT had little effect on the transactivating potential of this viral oncoprotein. Additional studies demonstrated that the association of mT with PLC-gamma1 was necessary and sufficient to activate both Ca2+- and Ras-dependent signaling cascades in Jurkat cells. These results indicate that PLC-gamma1 activation plays pivotal and pleiotropic roles in the stimulation of IL-2 gene expression, whereas activation of PI3-K negatively modulates this response in Jurkat T cells.

Protein-tyrosine kinase-dependent activation of STAT transcription factors in interleukin-2- or interleukin-4-stimulated T lymphocytes.

The proliferation of activated T lymphocytes is critically dependent on the binding of the T-cell growth factors, interleukin (IL)-2 and IL-4, to distinct but evolutionarily related cell surface receptors. Previous results suggest that the IL-2 receptor (IL-2R) and IL-4R are coupled to both overlapping and distinct intracellular signaling pathways in T lymphocytes. In this study, we demonstrate that activation of Janus tyrosine kinases (JAKs) and STAT transcription factors is rapidly induced by exposure of factor-dependent murine T-cell lines to IL-2 or IL-4. Both IL-2 and IL-4 stimulated the rapid activation of JAK1 and JAK3, whereas JAK2 activity was unaffected by either cytokine. These responses were accompanied by the appearance in cell nuclei of 3 DNA binding activities that recognized a high-affinity binding site for STAT factors. In transient transfection assays, this STAT factor target sequence conferred IL-2 and IL-4 inducibility on a synthetic luciferase reporter gene. Antibody supershifting experiments indicated that IL-2 induces the formation of STAT dimers containing STAT3 and STAT1 alpha. Although IL-4 also activated STAT1 alpha, the major IL4-induced STAT factor is not STAT3 and remains undefined. Pretreatment of the T-cells with the protein-tyrosine kinase inhibitor herbimycin A blocked both the nuclear translocation of STAT factors and STAT-dependent reporter gene transcription. Immunoblot analyses confirmed that cytoplasmic STAT3 was heavily phosphorylated on tyrosine in IL-2-stimulated cells, and that phosphorylated STAT3 appeared in the nuclei of these cells. These results indicate that identical JAKs and partially overlapping sets of STATs are activated by IL-2 and IL-4 in T lymphocytes.

Murine T helper cell-2 lymphocytes express type I and type II IL-1 receptors, but only the type I receptor mediates costimulatory activity.

The role of IL-1 in augmenting the Ag receptor-initiated activation program was evaluated in IL-4-producing (Th2) CD4+ murine T lymphocytes. Northern blot and 125I-labeled IL-1 alpha cross-linking analyses demonstrated that Th2 lymphocytes express both type I and type II IL-1R. The expression of both IL-1R isoforms on the surface of the Th2 cells is coordinately up-regulated in response to anti-CD3 cross-linking in the absence of detectable accessory cells. Analyses of the kinetics of IL-1R acquisition demonstrated that the peak level of type I and type II IL-1R mRNA expression occurs after the peak expression of mRNA encoding IL-2R alpha and IL-4, which are two IL-1-responsive events in the Th2 activation program. Type I IL-1R ligand-binding antagonists, IL-1R antagonist and anti-type I mAb, were used to evaluate the functional significance of Th2 cell expression of two IL-1R isoforms. The addition of either IL-1R antagonist or anti-type I mAb completely inhibited the IL-1 alpha-augmented component of the proliferative response stimulated by anti-CD3 plus exogenous IL-1 alpha. Together, these studies indicate that, although Th2 clones express inducible levels of both type I and type II IL-1R isoforms, the IL-1-induced intracellular signals involved in augmenting an anti-CD3-stimulated proliferative response are mediated solely through the type I IL-1R.

A beta polymorphic residues responsible for class II molecule recognition by alloreactive T cells.

In an effort to characterize the ligand that is bound by T helper lymphocyte antigen receptors, we have begun to identify class II polymorphic residues that comprise part of the allospecific TCR binding sites. Site-directed mutagenesis was used to construct mutant Ak beta (Ak beta*) genes that encode polypeptides into which single or multiple residues of the Ad beta polypeptide have been substituted in the beta 1 domain. A panel of cloned cell lines expressing the mutant Ak beta* Ak alpha or Ak beta* Ad alpha molecules was analyzed for the ability to stimulate Ak or Ad alloreactive T cell hybridomas. Substitution of d for k residues at specific positions in the beta 1 domain resulted not only in the loss of epitopes recognized by Ak-reactive T cells but, more importantly, in the gain of epitopes recognized by Ad-reactive T cells. Some of the polymorphic residues identified as contributing to the T cell epitopes are the same residues that contribute to the serologically immunodominant epitope. Other T cell epitopes map to positions predicted to be located either in an alpha-helix forming one side, or in a beta-pleated sheet forming the bottom of the putative antigen binding site. Thus, unlike serologic epitopes, TCR epitopes can be determined by A beta polymorphic residues in many different regions of the beta 1 domain and frequently depend upon contributions of A alpha polymorphic residues.

Regulation of murine MHC class II molecule expression. Identification of A beta residues responsible for allele-specific cell surface expression.

A panel of mutant class II genes have been constructed using site-directed mutagenesis and DNA-mediated gene transfer. Using this technique, Ak beta polypeptides have been altered by substituting one or more Ad beta-specific residues at polymorphic positions in the beta 1 domain. Transfection of M12.C3 B lymphoma cells with most mutant Ak beta* genes results in the expression of Ak beta* Ad alpha molecules on the cell surface. However, the substitution of a single d allele residue at position 78 or 86 in the Ak beta polypeptide results in either the complete absence or very low levels, respectively, of cell surface expression of the Ak beta* Ad alpha molecule, but does not alter Ak beta* Ak alpha expression. The T.86 Ak beta* Ad alpha is expressed primarily in an intracellular compartment while the T.78 Ak beta* molecule does not appear to be produced. The core-glycosylated T.78 Ak beta* polypeptide does, however, form a complex intracellularly with the core-glycosylated Ii polypeptide. Substitution of the combination of d allele residues at Ak beta polymorphic positions 9, 12, 13, 14, and 17 results in the absence of Ak beta* Ak alpha cell surface expression but does not alter the expression of this mutant Ak beta* polypeptide with the Ad alpha polypeptide. These allele-specific expression mutants demonstrate that substitution at certain beta 1 domain positions may result in the alteration of Ia cell surface expression and that the transport of Ia molecules from the Golgi apparatus to the cell surface may be regulated by signals that are determined by the interaction of polymorphic residues in both the alpha and beta polypeptides.

Identification of an immunodominant region on the I-A beta chain using site-directed mutagenesis and DNA-mediated gene transfer.

To identify which polymorphic residues determine the allospecific antibody binding sites on A beta polypeptides, mutant Ak beta genes were constructed encoding single or multiple amino acids of the d allele at 14 polymorphic positions in the beta 1 domain. Cell lines expressing these genes were analyzed by quantitative immunofluorescence using 16 mAbs reactive to Ak beta or Ad beta. Substitution of d allele residues at positions 63 and 65-67 in the Ak beta polypeptide resulted in the loss of binding of all Ak beta-reactive antibodies and the gain of binding of most Ad beta-reactive antibodies. Two Ad beta-reactive mAbs bound to the mutant Ak beta polypeptide containing d allele-characteristic residue at position 40. In contrast, substitution of the other polymorphic residues in the NH2-terminal and COOH-terminal regions of the beta 1 domain did not alter antibody binding.

DNA sequence analysis of I-Ak beta mutants reveals serologically immunodominant region.

We have produced a series of in vitro serologically selected cell lines that express mutant I-Ak molecules. In this report we describe the DNA sequence analysis of the Ak beta gene of four cell lines that express serologically altered Ak beta polypeptides in association with wild-type Ak alpha polypeptides. Each of the major serologic epitopes on the Ak beta polypeptide has been altered in one or more of the four mutants. In addition, the four mutants exhibit a broad spectrum of functional defects when used to stimulate a panel of T hybridomas of various specificities. The DNA sequence analysis revealed that each mutant had sustained a single nucleotide substitution resulting in a single amino acid substitution. All four independent substitutions occurred within or near the third of the four variable regions defined in the beta 1 domain of the A beta polypeptide by allelic comparisons. These data strongly suggest that the third variable region is the major determinant of alloantigenicity on the Ak beta polypeptide.

The appearance of liquid surfaces and layers in routine radiographs.

Image features in the radiograph produced by deformation of a liquid surface by surface tension and by the density gradient in a diffusion layer may present unexpected difficulty of interpretation. Such features have been analysed in model experiments, which have been reported earlier. The aim of the present investigation was to examine the occurrence and the clinical implications of corresponding phenomena in routine radiographs. In the human body liquid surfaces and diffusion layers can occur only in cavities, both normal and abnormal. A liquid surface tends to extend up a cavity wall to form a meniscoid or, if the cavity is small enough, a discoid. The liquid surface continues further up the wall as a liquid film. The shape of the meniscoid and the discoid varies with the shape and inclination of the wall. Most of the image features of interest are produced by rays that are tangential to a horizontal surface, a meniscoid, a discoid or a concave wall, any of which is visualized as an internal boundary with a light Mach line. When the wall is convex towards the cavity the meniscoid is saddle-shaped and an external boundary with a dark Mach line is produced. The horizontal part of a liquid surface can be touched only if it is at the same level as the focus of the roentgen tube. A liquid surface at any other level can be touched in its meniscoid only by rays that are not horizontal. It is reproduced as an internal boundary, slightly concave upwards; above this boundary the rest of the liquid surface is reproduced as a wedge field.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cell lines expressing mutant I-Ab and I-Ak molecules allows the definition of distinct serologic epitopes on A alpha and A beta polypeptides.

A series of seven I-Ab-reactive monoclonal antibodies (mAb) has been derived from a BALB/c anti-C57BL/6 immunization. Analysis of the reactivity patterns of these mAb with spleen cells of mice from the independent haplotypes has revealed three groups of mAb: group I mAb react with all haplotypes except d, group II with all except d and k, and group III with all except d, k, and j. In addition, the group I and group II mAb also react with human class II-expressing cells. We have used these mAb to isolate one mutant I-Ab-expressing cell line and three additional I-Ak mutant cell lines. These antibodies have been used, in conjunction with a large panel of I-A-reactive mAb available from others, to extensively characterize our collection of mutant I-A-expressing cell lines. Analysis of the mutant cell lines has allowed us to assign the reactivity of each mAb to either the A alpha- or the A beta-polypeptide. All seven newly isolated mAb appear to react with determinants on the A alpha-polypeptide. Furthermore, analysis of the panel of A alpha k- and A beta k-mutants has allowed us to discriminate at least five epitopes that are separable by mutation on the A beta k-polypeptide, and two epitopes on the A alpha k-polypeptide.

The layering phenomenon and boundary formation in radiographs.

In a radiographic examination using a water-soluble contrast medium this may form a layer beneath a body fluid. Between the two liquids a zone consisting of a mixture of the two liquids then forms through diffusion. This diffusion layer produces some characteristic features in the radiographic image, an analysis of which was the purpose of the model experiments performed in this investigation. In this analysis of the layering phenomenon the radiographed objects were cylindrical tubes of methyl methacrylate partly filled with water. In some cases a rod was placed concentrically in the tube. Contrast medium was layered below the water. Radiographs were produced with the tube either vertical or inclined, and with either a horizontal or a vertical projection. In the image the layer of contrast medium was visualized as a light field, and the water layer as an overlying relatively dark field. The diffusion layer was visualized as a transitional zone--the diffusion field. Distinct boundaries and Mach lines observed in the bottom field were produced by the interface between the contrast medium and the solid wall where it was touched by the roentgen rays. These boundaries continued into the diffusion field where they gradually became less visible and eventually disappeared. The upper and lower boundaries of the diffusion field were diffuse and associated with dark and light Mach bands, respectively. The upper boundary appeared to be convex upwards. In the case of the inclined model and a vertical beam the diffusion field was elliptical, with a still more diffuse transition to the fields above and below than in the case of the vertical model and a horizontal beam.(ABSTRACT TRUNCATED AT 250 WORDS)

Function of human autologous kidney grafts after extracorporeal preservation with Sacks' II solution.

The function of 18 human kidney grafts was studied before and 3 months after autotransplantation without prior bench surgery on the parenchyma or the vessels. Kidney protection was accomplished by pre-treating the patient with mannitol and low molecular weight dextran solution and flushing the kidney with a Xylocaine-heparin mixture and cold Sacks' II solution. The mean cold ischaemia time was 239 min (range 140-345 min). The glomerular filtration rate (GFR) remained unchanged post-operatively in terms of both total and split kidney GFR. Proximal and distal tubular integrity, as studied by determination of beta2-microglobulin excretion and concentration ability respectively, was also preserved.

Radiographic demonstration of a liquid surface bounded by a wet solid surface.

At the line of contact between a liquid surface and a bounding wet solid wall deformation of the surface is produced by surface tension, and this deformation gives rise to various features in the radiograph. For the interpretation of these image features a detailed analysis was performed by means of model experiments. Tubes and rods of methyl acrylate with different diameters and covered with wet sheets of porous paper were used. The tubes were partly filled with water. In some cases a rod was placed concentrically or eccentrically inside the tube. The shape of the image of the liquid surface in vertical and inclined models was examined by conventional radiographic technique and computed tomography. At the walls the surface tension resulted in formation of meniscoids. When the distance between solid walls was short enough adjacent meniscoids joined to form a discoid. The shape of the meniscoid and discoid varied with the shape and inclination of the walls. Distinct boundaries representative of the shape and position of the surface were produced only from those places where the beam was tangential to the surface. When the surface was concave towards the gas in the direction of the beam an internal boundary with a light Mach line was produced. When the surface was convex towards the gas in the direction of the beam an external boundary with a dark Mach line was produced. A boundary was straight when the surface was plane or was part of a cylindric surface. The seemingly simple form of the liquid surface gave rise to surprisingly complex boundary formations, which are analyzed in detail. The findings reported may have a variety of implications for the interpretation of clinical radiographs and may act as a key for the reconstruction of the structures reproduced.

Urodynamic consequences of a direct pyelocystostomy at autotransplantation of the kidney.

The urodynamic consequences of renal autotransplantation and pyelocystostomy were studied in 17 male and 3 female patients. The indications of the procedure were urothelial tumor of the upper urinary tract, remaining outflow obstruction after conventional pyeloplasty and recurrent stones. The mean observation time was 31 months (range 3-50 months). The micturition flow rate was unchanged. There was an insignificant increase in residual urine. No evidence of any clinically significant disturbance of the sensory or motor bladder innervation or reduction of the functional bladder capacity was found. Urethrocystography showed transient dilatation of calyces at micturition. Urinary tract infections, when present, had the same pattern as preoperatively. Thus, autotransplantation of the kidney with a wide, direct pyelocystostomy is consistent with essentially normal urodynamics of the transplanted renal pelvis and the bladder.

Natural history of renal artery aneurysm elucidated by repeated angiography and pathoanatomical studies.

The findings from repeated angiographies in 16 female and 5 male patients with altogether 34 renal artery aneurysms were studied. The mean interval between the first and last angiography was 35 months. Seven patients had multiple aneurysms. Two to four angiographies were performed in each patient. They showed no change in 28 aneurysms and slight or minimal enlargement, thrombosis or calcification in the other 6. The clinical course was uneventful except for severe hypertension in 3 patients. No rupture occurred. Eight patients, of whom 5 had solitary, saccular aneurysms, were operated upon. Pathoanatomically, fibromuscular dysplasia or secondarily changed fibromuscular dysplasia was found in 7 of them. Four died of unrelated disease having been followed up for 55-204 months (mean 102 months). Nine were alive and symptomless at the end of follow-up 11-195 months (mean 97 months) after the first angiography. The study supports the view that the risk of rupture of a renal artery aneurysm is very small, and indicates that fibromuscular dysplasia is common even when the angiography shows solitary, saccular aneurysm only.

The structure-function relationship of I-A molecules: correlation of serologic and functional phenotypes of four I-Ak mutant cell lines.

We have isolated and characterized four mutant I-Ak-expressing cell lines derived from the B cell-B lymphoma hybrid antigen-presenting cell line TA3. The mutants were isolated by first selecting against expression of one Ak epitope by treatment with a monoclonal antibody in the presence of complement and then selecting for retention of a second Ak epitope by electronic cell-sorting of cells stained for fluorescence with a second monoclonal antibody. The serologic and functional phenotypes of the mutants were characterized by using panels of I-Ak-specific monoclonal antibodies and I-Ak-restricted T hybridomas. We obtained one Ak alpha mutant (J4) that no longer reacts with any Ak alpha-specific antibody and also is incapable of stimulating any I-Ak-restricted T hybridoma. We obtained three Ak beta mutants (LD3, K5, G1) that express a wide range of serologic and functional phenotypes. Correlation of the serologic and functional phenotypes reveals that the serologic epitope Ia.1 may overlap with a major site of T cell recognition, whereas the Ia.17 serologic epitope appears to be only a minor site for T cell recognition.

Treatment of urothelial tumors of the upper urinary tract by nephroureterectomy, renal autotransplantation, and pyelocystostomy.

Nephroureterectomy, renal autotransplantation, and pyelocystostomy have been performed in eight patients with urothelial tumors of the upper urinary tract. One patient had tumors in a solitary kidney, two patients had bilateral tumors, and five patients had unilateral tumors. Three patients have had recurrent calyceal tumors which were successively managed by the transurethral route. In one patient the kidney had to be removed after 4.5 years because of infiltrating tumor recurrence. Two patients died; the renal pelvis of the graft was tumor free at autopsy in both cases. The other five patients are alive and free from tumor recurrence. The procedure implies increased radicality compared with conventional conservative treatment and simplified follow-up. It may be considered in patients with bilateral tumors or tumors of a solitary kidney, and in selected patients with unilateral low-grade, low-stage tumors.

Widespread urothelial tumor treated with bilateral nephroureterectomy, cystectomy, autotransplantation of one kidney and pelvioileocutaneostomy.

We report on a man who had recurrent low grade noninvasive bladder tumors for 8 years and an increasing number of such tumors in both renal pelves and ureters for 3 years. Because of steady tumor progression, including obstruction of urine outflow from the right kidney, the tumors and as much urothelium as possible were removed. Right nephroureterectomy was performed initially. A month later left nephroureterectomy, cystectomy, extracorporeal pelvic resection and autotransplantation of the kidney to the pelvic cavity with urinary diversion to the skin using an isolated ileal loop were done. Convalescence was uneventful. Endoscopic examination 4 months postoperatively via the ileal conduit revealed no recurrences. The general condition of the patient after 6 months was excellent.