Sex dimorphic actions of rosiglitazone in generalised peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-deficient mice.

AIMS/HYPOTHESIS: The aim of this study was to determine the dependency on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) of insulin sensitisation and glucose homeostasis by thiazolidinediones using a global Ppar-gamma (also known as Pparg)-knockout mouse model. METHODS: Global Mox2-Cre-Ppar-gamma-knockout (MORE-PGKO) mice were treated with rosiglitazone and analysed for insulin sensitivity and glucose metabolism. Metabolic and hormonal variables were determined. Adipose and other tissues were measured and analysed for gene expression. RESULTS: Rosiglitazone induced regrowth of fat in female but not male MORE-PGKO mice, and only in specific depots. Insulin sensitivity increased but, surprisingly, was not associated with the typical changes in adipokines, plasma NEFA or tissue triacylglycerol. However, increases in alternatively activated macrophage markers, which have been previously associated with metabolic improvement, were observed in the regrown fat. Rosiglitazone improved glucose homeostasis but not insulin sensitivity in male MORE-PGKO mice, with further increase of insulin associated with an apparent expansion of pancreatic islets. CONCLUSIONS/INTERPRETATION: Stimulating fat growth by rosiglitazone is sufficient to improve insulin sensitivity in female mice with 95% PPAR-gamma deficiency. This increase in insulin sensitivity is not likely to be due to changes typically seen in adipokines or lipids but may involve changes in macrophage polarisation that occur independent of PPAR-gamma. In contrast, rosiglitazone improves glucose homeostasis in male mice with similar PPAR-gamma deficiency by increasing insulin production independent of changes in adiposity. Further, the insulin-sensitising effect of rosiglitazone is dependent on PPAR-gamma in this male lipodystrophic model.

PPARgamma-dependent and PPARgamma-independent effects on the development of adipose cells from embryonic stem cells.

Peroxisome proliferator-activated receptor (PPAR) gamma was shown to be required for adipocyte formation both in vivo and in vitro. However, the role of PPARgamma in the initial steps of adipose cell development was not distinguished from its role in the terminal steps. We now show that PPARgamma is expressed early in embryoid bodies (EBs) derived from embryonic stem cells and in E.8.5 mouse embryos. Addition of a specific ligand for PPARgamma in developing EBs over-expressing PPARgamma did not commit stem cells towards the adipose lineage. In differentiated PPARgamma(-/-) EBs, only markers characteristic of preadipocytes were found to be expressed. PPARdelta is present in EBs but did not compensate for the lack of PPARgamma in terminal differentiation. Taken together, these results favor a critical PPARgamma-independent phase culminating in preadipocyte formation that precedes a PPARgamma-dependent phase in the development of adipose cells from pluripotent stem cells.

Impaired activation of murine platelets lacking G alpha(i2).

The intracellular signaling pathways by which G protein-coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the G(i) pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet G alpha(i) subtype, G alpha(i2), has been disrupted. In intact platelets from G alpha(i2)-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin alpha IIb beta 3 (GPIIb-IIIa) were strongly reduced in platelets from G alpha(i2)-deficient mice. In addition, G alpha(i2)-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase--coupled platelet ADP receptor (P2Y(12)) on wild-type platelets with a selective antagonist. These observations suggest that G alpha(i2) is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the G alpha(i2)-linked ADP receptor.

A major role for VCAM-1, but not ICAM-1, in early atherosclerosis.

VCAM-1 and ICAM-1 are endothelial adhesion molecules of the Ig gene superfamily that may participate in atherogenesis by promoting monocyte accumulation in the arterial intima. Both are expressed in regions predisposed to atherosclerosis and at the periphery of established lesions, while ICAM-1 is also expressed more broadly. To evaluate functions of VCAM-1 in chronic disease, we disrupted its fourth Ig domain, producing the murine Vcam1(D4D) allele. VCAM-1(D4D) mRNA and protein were reduced to 2-8% of wild-type allele (Vcam1(+)) levels but were sufficient to partially rescue the lethal phenotype of VCAM-1-null embryos. After crossing into the LDL receptor-null background, Vcam1(+/+) and Vcam1(D4D/D4D) paired littermates were generated from heterozygous intercrosses and fed a cholesterol-enriched diet for 8 weeks. The area of early atherosclerotic lesions in the aorta, quantified by en face oil red O staining, was reduced significantly in Vcam1(D4D/D4D) mice, although cholesterol levels, lipoprotein profiles, and numbers of circulating leukocytes were comparable to wild-type. In contrast, deficiency of ICAM-1 either alone or in combination with VCAM-1 deficiency did not alter nascent lesion formation. Therefore, although expression of both VCAM-1 and ICAM-1 is upregulated in atherosclerotic lesions, our data indicate that VCAM-1 plays a dominant role in the initiation of atherosclerosis.

Targeted inactivation of Galpha(i) does not alter cardiac function or beta-adrenergic sensitivity.

Inhibitory Galpha(i) protein increases in the myocardium during hypertrophy and has been associated with beta-adrenergic receptor (beta-AR) desensitization, contractile dysfunction, and progression of cardiac disease. The role of Galpha(i) proteins in mediating basal cardiac function and beta-AR response in nonpathological myocardium, however, is uncertain. Transgenic mice with targeted inactivation of Galpha(i2) or Galpha(i3) were examined for in vivo cardiac function with the use of conscious echocardiography and for ex vivo cardiac response to inotropic stimulation with the use of Langendorff blood-perfused isolated hearts and adult ventricular cardiomyocytes. Echocardiography revealed that percent fractional shortening and heart rate were similar among wild-type, Galpha(i2)-null, and Galpha(i3)-null mice. Comparable baseline diastolic and contractile performance was also observed in isolated hearts and isolated ventricular myocytes from wild-type mice and mice lacking Galpha(i) proteins. Isoproterenol infusion enhanced diastolic and contractile performance to a similar degree in wild-type, Galpha(i2)-null, and Galpha(i3)-null mice. These data demonstrate no observable role for inhibitory G proteins in mediating basal cardiac function or sensitivity to beta-AR stimulation in nonpathological myocardium.

The role of PPAR-gamma in macrophage differentiation and cholesterol uptake.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), the transcription factor target of the anti-diabetic thiazolidinedione (TZD) drugs, is reported to mediate macrophage differentiation and inflammatory responses. Using PPAR-gamma-deficient stem cells, we demonstrate that PPAR-gamma is neither essential for myeloid development, nor for such mature macrophage functions as phagocytosis and inflammatory cytokine production. PPAR-gamma is required for basal expression of CD36, but not for expression of the other major scavenger receptor responsible for uptake of modified lipoproteins, SR-A. In wild-type macrophages, TZD treatment divergently regulated CD36 and class A macrophage-scavenger receptor expression and failed to induce significant cellular cholesterol accumulation, indicating that TZDs may not exacerbate macrophage foam-cell formation.

Galpha(i2) but not Galpha(i3) is required for muscarinic inhibition of contractility and calcium currents in adult cardiomyocytes.

Parasympathetic stimulation of the heart acts through M(2)-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins Galpha(i/o), the specific signal transduction requirements of Galpha(i2) and Galpha(i3) in mediating muscarinic regulated L-type calcium currents (I(Ca, L)), intracellular calcium, and cell contractility remain to be determined. Adult ventricular myocytes were isolated from Galpha(i2)-null mice, Galpha(i3)-null mice, and their wild-type littermates. Cell shortening, intracellular calcium levels, and I(Ca, L) were all measured in response to isoproterenol, a beta-adrenergic receptor agonist, and carbachol, a cholinergic receptor agonist. With isoproterenol stimulation, myocytes from all groups demonstrated a marked increase in calcium currents, correlating with augmented intracellular calcium transient amplitude and cell shortening. Carbachol significantly attenuated the isoproterenol response in wild-type and Galpha(i3)-null cells but had no effect in Galpha(i2)-null cells. This study demonstrates that Galpha(i2), but not Galpha(i3), is required for muscarinic inhibition of the beta-adrenergic response in adult murine ventricular myocytes.

E-selectin expression and function in a unique placental trophoblast population at the fetal-maternal interface: regulation by a trophoblast-restricted transcriptional mechanism conserved between humans and mice.

Trophoblast are the earliest differentiated cells to emerge during mammalian ontogeny. Proper differentiation and maturation of trophoblast contributes to the fetal-maternal vascular interface of the mature placenta and is required for all subsequent stages of embryogenesis. Although lineage commitment and early differentiation of trophoblast have been investigated experimentally, molecular markers and regulatory mechanisms operating later in trophoblast development remain uncertain. We now report that E-selectin is expressed in a unique pattern in secondary trophoblast giant cells, trophoblast lining the central artery, and a subpopulation of labyrinthine trophoblast all located at the fetal-maternal interface of the murine placenta. These cells line vascular channels but express a unique profile of gene products not displayed by vascular endothelium. Placentae lacking E-selectin show increased trophoblast glycogen cells and fewer labyrinthine neutrophils compared with normal placentae, suggesting that recognition of E-selectin on trophoblast by counter-receptors on other cells contributes to placental development. Novel, distant first exons direct E-selectin expression in both murine and human placentae, suggesting that evolutionarily conserved and lineage-restricted transcriptional mechanisms regulate expression in homologous trophoblast populations in both species. These results define, at molecular and anatomic levels, a unique population of trophoblast located at the physiologically critical fetal-maternal vascular interface in mice. We also present initial functional characterization of E-selectin in placenta. These results support the general hypothesis that endothelial-leukocyte adhesion molecules performing specialized functions in adults may also function in development of human and murine hemochorial placentae.

E-selectin expression and stimulation by inflammatory mediators are developmentally regulated during embryogenesis.

Leukocyte recruitment during inflammation is specified, in part, by the spatial distribution and temporal regulation of endothelial adhesion molecules. In this study we investigated the developmental onset of E-selectin and intercellular adhesion molecule-1 (ICAM-1) basal expression and inducibility by inflammatory mediators as indices of lineage-restricted endothelial adhesion molecule expression. We studied both murine embryos and embryoid bodies (EB), derived from differentiated embryonic stem cells, to examine a broad range of endothelial ontogeny. Our results reveal that E-selectin and ICAM-1 are differentially regulated during development and that three stages define the ontogeny of the E-selectin-inducible response. The earliest endothelial lineage cells in Day 4 and Day 5 EB did not express E-selectin in the basal state or after stimulation. A second stage, observed between embryonic Day 9.5 (E9.5) and E11.5 to E12.5 in cultured embryo cells and transiently at Day 6 of EB differentiation, was characterized by basal expression that was not stimulated by inflammatory mediators. A third stage was characterized by both basal and inducible expression of E-selectin and was observed beginning at E12.5 to E13.5 in cultured embryo cells and at Day 7 in EB. In contrast ICAM-1 was stimulated at all of the embryonic stages examined and before the onset of E-selectin inducibility in both embryos and EB. E-selectin expression in embryos was also stimulated by introducing endotoxin into the embryonic, but not the maternal, peritoneum. This suggests that embryos are protected from inflammatory insults present in the maternal circulation. The developmentally regulated acquisition of E-selectin inducibility during embryogenesis likely involves changes in signal transduction cascades, transcription factors, and/or chromatin accessibility that specify inducible expression within the endothelial lineage and further restrict inducibility to particular endothelial subpopulations.

PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro.

The process of adipogenesis is known to involve the interplay of several transcription factors. Activation of one of these factors, the nuclear hormone receptor PPAR gamma, is known to promote fat cell differentiation in vitro. Whether PPAR gamma is required for this process in vivo has remained an open question because a viable loss-of-function model for PPAR gamma has been lacking. We demonstrate here that mice chimeric for wild-type and PPAR gamma null cells show little or no contribution of null cells to adipose tissue, whereas most other organs examined do not require PPAR gamma for proper development. In vitro, the differentiation of ES cells into fat is shown to be dependent on PPAR gamma gene dosage. These data provide direct evidence that PPAR gamma is essential for the formation of fat.

Galpha(i2), Galpha(i3)and Galpha(o) are all required for normal muscarinic inhibition of the cardiac calcium channels in nodal/atrial-like cultured cardiocytes.

The cardiac L-type calcium current (I(Ca,L)) is an important regulator of myocardial contractility. It is activated by sympathetic stimulation and inhibited by parasympathetic activity via muscarinic acetylcholine receptors. Muscarinic inhibition of I(Ca,L) occurs via activation of pertussis toxin (PTX)-sensitive heterotrimeric G-proteins. Although recent studies have shown that expression of G(oalpha) is important for this effect in adult mouse ventricular cells, two other PTX-sensitive G-proteins (G(i2) and G(i3)) are also expressed in cardiocytes and are activated. Their role in the regulation of I(Ca,L) has not been examined. In addition, it is not known whether nodal/atrial cardiac cells use the same G-proteins. We show that gene inactivation of each of the three PTX-sensitive Galpha-proteins (alpha(i2), alpha(i3), and alpha(o)) affects muscarinic inhibition of cardiac I(Ca,L) in embryonic stem (ES) cell-derived cardiocytes. Inactivation of either alpha(i2) or alpha(i3) markedly slows the time course of muscarinic inhibition of I(Ca,L), and in cells where both alpha(i2) and alpha(i3) are inactivated the effects are not additive. We also establish an essential role for alpha(o)in this atrial/nodal-like cardiocyte system and show that alpha(o)acts proximal to NO generation. NO generation plays a critical role in I(Ca,L) regulation since the nitric oxide synthase (NOS) antagonist, l -NMMA, blocked the inhibition of I(Ca,L) in WT and in alpha(i2)/alpha(i3)-null cells. In WT cells, the NO generating agent SIN-1 inhibited I(Ca,L) and the addition of carbachol resulted in faster inhibition, suggesting that pathways in addition to NO are also activated. This study shows that alpha(i2) and alpha(i3) play a critical role in the normal inhibition of cardiocyte I(Ca,L). Thus, all muscarinic receptor activated G-proteins (G(i2), G(i3) and G(o)) are necessary for normal inhibition and act through both NO and non-NO signaling pathways.

Simultaneous Cre catalyzed recombination of two alleles to restore neomycin sensitivity and facilitate homozygous mutations.

Cells homozygous for neo-expressing mutations can be derived by culturing heterozygotes with elevated G418. We demonstrate that this strategy is significantly less efficient if hyg is substituted for neo. Therefore, to introduce additional mutations Cre recombinase was used to remove floxed neo from both alleles of homozygotes at two different loci. The rate-determining step in Cre excision appeared independent of substrate copy number. Incorporating cytosine deaminase and Herpes simplex virus thymidine kinase allowed negative selection for both targeting and Cre excision. The resulting G418-sensitive homozygous mutants should allow mutagenesis at additional loci and avoid untoward effects of retained selection markers.

Mice lacking E-selectin show normal numbers of rolling leukocytes but reduced leukocyte stable arrest on cytokine-activated microvascular endothelium.

OBJECTIVE: Previous work indicated that E-selectin mediates transient interactions between leukocytes and cytokine-activated endothelium in vitro. Here we examine the role of E-selectin in blood leukocyte interactions with microvascular endothelium in vivo. METHODS: E-selectin-deficient (E-/-) mice were produced by gene targeting. The effect of this null mutation on leukocyte-endothelial interactions was determined by intravital microscopy before and 4 to 5 hours after local administration of the proinflammatory cytokine tumor necrosis factor alpha (TNF alpha) in dermal microvessels with low blood flow (dorsal skin-fold chambers, intact ear skin), and after endotoxin activation in exteriorized mesenteric microvessels with higher blood flow. RESULTS: E-/- mice were viable, fertile with normal circulating leukocyte and platelet profiles. Approximately 60% of circulating leukocytes rolled in dermal microvessels of both normal (E+/+) and E-/- mice without inflammatory stimulation. After local administration of TNF alpha, rolling increased modestly and equivalently in both genotypes. The main effect of TNF alpha was a dramatic increase in leukocyte stable adhesion and, unlike rolling, this manifestation of endothelial activation was significantly reduced in E-/- animals. This reflected fewer dermal microvessels supporting higher adhesion densities in E-/- mice, and a similar trend was observed in mesenteric microvessels. CONCLUSIONS: E-selectin plays a previously unappreciated role in facilitating and/or mediating stable adhesion of leukocytes to inflamed microvascular endothelium.

Microvascular endothelial cells from E-selectin-deficient mice form tubes in vitro.

Studies of capillary morphogenesis and angiogenesis in vitro have suggested a role for E-selectin (CD62E) in the process of differentiation into tube-like structures. Recent studies by our group and others have demonstrated that mice lacking E-selectin because of germline inactivation of the E-selectin gene by gene targeting are viable and fertile, without apparent deficiencies in vascular development. Murine lung endothelial cells from wild-type and E-selectin-deficient animals were isolated using an activation-dependent sterile sorting method, and differentiation into tube-like structures on sparse fibronectin, Matrigel, and collagen gels was compared. Both types of murine lung endothelial cells spontaneously organized to form multicellular tubes and extensive anastomotic networks. There were no major differences in either the time course of development or the general appearance of the multicellular cords or tube-like structures formed by murine lung endothelial cells from wild-type or E-selectin-deficient mice, although different patterns were observed on different extracellular matrices. These studies, thus, demonstrate that E-selectin is not required for morphogenesis of 3-dimensional vascular structures in vitro.

Activation-dependent isolation and culture of murine pulmonary microvascular endothelium.

OBJECTIVE: Establish a reproducible method for the isolation and cultivation of murine pulmonary microvascular endothelium. To this end, we exploited the localized pattern of microvascular endothelial activation induced in vivo by inflammatory stimuli to isolate a subpopulation of endothelium for in vitro study. METHODS: Immunohistochemical analyses of the pulmonary vasculature of mice treated systemically with gram-negative bacterial endotoxin (LPS) demonstrated selective expression of VCAM-1 (CD106) in the endothelial lining of small collecting veins, venules, septal capillaries, and, infrequently, small arteries, which was not observed in control mice. Single cell suspensions prepared by enzymatic dissociation of peripheral lobular tissues dissected from the lungs of LPS-stimulated mice were incubated with a phycoerythrin-conjugated antimouse VCAM-1 monoclonal antibody (MK 1.91). Cells expressing this antigen were isolated by sterile fluorescence-activated cell sorting (FACS). Positive cell populations were collected and cultured for 1-2 weeks. When confluent, these primary cultures were further FACS enriched for endothelium, positively selecting for cells incorporating a fluorescent derivative of acetylated low density lipoprotein (Di-I-Ac-LDL). RESULTS: The resulting population of cells (mouse lung endothelial cells, MLEC) were uniformly positive for the endothelial markers von Willebrand factor, thrombomodulin, and Dil-Ac-LDL uptake. MLEC readily formed tube-like structures when cultured on Matrigel and spontaneously demonstrated a sprouting phenotype on fibronectin or collagen matrices. MLEC retained responsiveness to cytokines (IL-1 alpha, IL-1 beta, TNF alpha, IFN gamma) up to at least eight passages from primary culture and demonstrated upregulation of E-selectin (CD62E) and P-selectin (CD62P) mRNA as early as 2 hr after LPS stimulation. Characteristic temporal expression patterns of cell surface E-selectin (maximal at 4 hr and declining toward baseline by 24 hr), VCAM-1 (maximal at 6-8 hr and remaining elevated for 24-48 hr), and ICAM-1 (maximal at 6-8 hr and maintained at 24 hr) were observed when cultured MLEC were treated with recombinant murine TNF alpha or recombinant human (rh) IL-1 alpha or rhIL-1 beta. The rolling, adhesion, and transmigration of human polymorphonuclear leukocytes was markedly increased on cytokine-activated MLEC monolayers under defined flow conditions. CONCLUSION: The strategy of activation-dependent isolation allows for the reproducible selection of a specific subset of microvascular endothelial cells for in vitro study. This experimental approach should further facilitate study of the functional heterogeneity of endothelium and its pathophysiologic dysfunction.

Flagellar and acrosomal abnormalities associated with testicular HSV-tk expression in the mouse.

Expression of herpes simplex virus thymidine kinase (HSV-tk) in transgenic mouse testis is associated with abnormalities in spermatogenesis leading to infertility. Our studies of this phenomenon in two transgenic lines led to the identification of a genetic locus that reduced testicular HSV-tk activity and restored fertility. Using light and electron microscopy, we examined spermatogenesis in the infertile transgenic males as well as in the fertile revertants. Infertile males from line 21OH1 had high levels of testicular HSV-tk activity, acrosomal aberrations, and a developmental arrest in spermatogenesis. Infertile males from line ANF1 had lower levels of testicular HSV-tk expression and demonstrated a unique set of structural changes present in the neck and flagellum of epididymal sperm. Revertant ANF1 males, with a significant decrease in testicular HSV-tk expression and a restoration of fertility, showed a marked reduction in the number of sperm abnormalities. Several of the ANF1-specific abnormalities were similar to lesions reported in the sperm of mouse t locus mutants, mouse wobbler homozygotes, and bulls with the Dag-defect.

An element regulating adrenal-specific steroid 21-hydroxylase expression is located within the slp gene.

In this report we demonstrate that a transcriptional regulatory element for one gene lies within a second, seemingly unrelated gene. Specifically, the 3' portion of the murine sex-limited protein (slp) gene, located within the class III region of the major histocompatibility complex, contains an element that regulates expression of the linked steroid 21-hydroxylase gene. A 4.2-kilobase (kb) major histocompatibility complex region, located between -2.2 and -6.4 kb upstream of 21OH-A, is required for expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Two short regions of DNA, located between -5.3 and -6.0 kb, stimulate chloramphenicol acetyltransferase expression in Y1 adrenocortical tumor cells, and both of these active regions lie within the slp gene. A 21-base pair sequence, which is required for activity of the most 3' region, does not contain any of over 100 previously identified transcriptional regulatory elements. This juxtaposition of structural and regulatory elements of otherwise unrelated genes suggests a mechanism by which the evolutionarily conserved genetic linkage of 21OH-A and slp (or the homologous complement component C4) might provide a selective advantage. Analogous genetic arrangements may explain other examples of conserved linkage of disparate genes.

cis modification of the steroid 21-hydroxylase gene prevents its expression in the Y1 mouse adrenocortical tumor cell line.

The Y1 mouse adrenocortical tumor cell line retains the ability to synthesize and secrete steroids, but does not express steroid 21-hydroxylase (C21) and, therefore, does not produce 21-hydroxylated steroids. In this investigation the mechanisms underlying the loss of C21 activity in the Y1 cell line were explored. A 9-kilobase BglII fragment containing the C21 gene was cloned from the Y1 genome. This genomic clone directed the synthesis of C21 transcripts and 21-hydroxylated steroid products when transfected back into the Y1 cell line. As determined by restriction endonuclease digestions with MspI and HpaII, enzymes that distinguish between unmethylated and methylated CCGG sites, the endogenous C21 gene was extensively methylated in Y1 adrenal cells and in cells from other mouse tissues that do not normally express this gene. In contrast, the C21 gene was hypomethylated in primary cultures of mouse adrenal cells which normally synthesize large amounts of C21. The cloned C21 gene transfected into Y1 cells initially was unmethylated, but became extensively methylated with prolonged culture of the cells; prolonged culture of these transfectants also resulted in a loss of C21 expression. Loss of C21 expression in Y1 transfectants, however, temporally preceded the extensive methylation of the transfected C21 gene. Furthermore, treatment of Y1 cells with 5-azacytidine caused a demethylation of the endogenous C21 gene, but did not result in the recovery of C21 expression. These results indicate that Y1 cells contain a functional C21 gene that has been silenced by a reversible cis-modification event.(ABSTRACT TRUNCATED AT 250 WORDS)

The glycoprotein nature and biosynthetic relationship of two molecularly distinct species of primate T-cell growth factor.

T-cell growth factor (TCGF) produced by the MLA 144 gibbon ape T-lymphosarcoma cell line was biosynthetically radiolabelled with [35S]methionine and isolated by reverse-phase h.p.l.c. [Milstone & Parker (1983) Biochem. Biophys. Res. Commun. 115, 762-768]. Two predominant species, of Mr 16300 and pI 6.8 and of Mr 14300 and pI 7.5, were resolved. Analysis of TCGF labelled with a mixture of [3H]glucosamine and [14C]methionine demonstrated that only the 16300-Mr protein contained detectable carbohydrate label, approx. 50% of which was present in sialic acid residues, which were largely responsible for the charge difference observed between the two forms of TCGF. The 14300-Mr protein was labelled within 5 min after addition of [35S]methionine and was the predominant intracellular form of TCGF, whereas the 16300-Mr protein was not detected until 25 min later and was the predominant extracellular form of TCGF. Pulse-chase experiments were consistent with the hypothesis that the 14300-Mr protein is an intracellular precursor that is glycosylated to form the 16300-Mr protein, which is then preferentially secreted from the cell.

Interleukin 2-mediated induction of lytic activity in a cloned murine CTL line.

We have analyzed the ability of interleukin 2 (IL 2) to induce lytic activity within a cloned murine H-2Dd-specific CTL line. Weakly lytic CTL harvested 6 to 7 days after previous stimulation with irradiated DBA/2J spleen cells and conditioned medium from secondary MLC (MLC SN) could be reactivated to high antigen-specific lytic activity with highly purified gibbon IL 2 or E. coli-produced human recombinant DNA IL 2. Dose-response curves with IL 2 and MLC SN suggest that IL 2 may be the principal detectable activity in MLC SN that is active on these CTL. Doses of IL 2 or MLC SN that were saturating for the induction of lytic activity were suboptimal for the expression of DNA synthesis measured by 3HTdR incorporation. This is consistent with a mechanism in which different threshold IL 2 concentrations are required to induce these two biologic responses. Finally, we show that IFN-gamma has little effect on the expression of lytic activity either alone or in combination with IL 2 in this bioassay.