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J S Hunt

Publications and source records attributed to J S Hunt.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification and cellular localization of unique interferon mRNA from human placenta.

Although constitutive expression of trophoblast or pregnancy-associated interferon (IFN) has long been recognized, their cDNA sequences have been determined for only ruminant ungulates. Here we show a human trophoblast IFN (htIFN) cDNA whose nucleotide sequence is very similar (85% identity) to that of ovine and bovine trophoblast IFNs, IFN tau s. Like ruminant IFN tau s, htIFN cDNAs contain an open reading frame of 195 codons including a signal sequence of 23 amino acids, resulting in a mature polypeptide of 172 amino acids. The deduced amino acid sequence of htIFN shares 73, 62, and 56% identities with ovine IFN tau, human IFN alpha II, and human IFN alpha I, respectively. However, the expression of htIFN is not limited to a specific period of pregnancy because transcripts of htIFN genes are detected in human lymphocytes, cells obtained by amniocentesis (amniocytes), first trimester, and term placentas. Human trophoblast IFN mRNA is localized mainly in extravillous trophoblasts cells of placental villi, particularly in the migrating cytotrophoblasts cells, which eventually replace maternal endothelial cells in spiral arteries of the decidua. Both sense and antisense mRNAs for human IFN alpha II are localized in the outer layer of villous structures. Coexistence of these mRNAs at the placental villi throughout pregnancy suggests that, in addition to a role in placental cell growth and differentiation, IFNs may play a role protecting the fetus in viral environments.

Amino Acid Sequence↗
PubMed1994

Gestation-related expression of the interferon-gamma receptor gene in mouse uterine and embryonic hematopoietic cells.

Macrophages and natural killer (NK)-like cells are the major hematopoietic cell populations in the cycling and pregnant mouse uterus and are also found in the embryo. In order to evaluate potential receptivity of these cells to interferon-gamma (IFN-gamma), tissues taken from cycling and pregnant mice were tested for IFN-gamma receptor (IFN-gamma R) mRNA and protein. Macrophages were identified immunohistochemically by using the specific monoclonal antibody F4/80. NK cells were identified by their large size, distinctive intracellular granules, and binding of a monoclonal antibody to the common leukocyte antigen. In cycling uteri, the abundance of IFN-gamma R mRNA relative to an invariant message (glyceraldehyde-3-phosphate dehydrogenase) increased during progression of the hormonally regulated estrous cycle. IFN-gamma R mRNA in situ hybridization signals were slightly higher in macrophage-like than in other types of endometrial stromal cells. In pregnant uteri, the highest proportions of IFN-gamma R mRNA were observed at gestation day (g.d.) 16. Specific message and protein were present in uterine macrophages by g.d. 7 and in NK cells by g.d. 9. IFN-gamma R expression in both lineages remained stable through the balance of pregnancy. In embryos, IFN-gamma R mRNA increased between g.d. 14 and 16. Specific transcripts were present in many cells at g.d. 14, but none were detected in embryonic liver macrophages until g.d. 16. The results of this study suggest relationships between IFN-gamma R expression and ovarian hormones as well as cell maturation and support the postulate that IFN-gamma receptor-ligand interactions may improve the ability of uterine and embryonic hematopoietic cells to perform specific tasks during gestation.

Animals↗
PubMed1994

Tumor necrosis factor-alpha mRNA and protein in endometrial tumors: analysis by in situ hybridization and immunocytochemistry.

Abnormal expression of polypeptide growth factors and their receptors is closely associated with tumorigenic transformation. In this study tumor necrosis factor-alpha (TNF-alpha) mRNA and protein were analyzed in polyps and proliferative lesions of endometrium as well as in low and high grade endometrial tumors by using in situ hybridization and immunocytochemistry. All samples contained products of the TNF-alpha gene. Histochemical scores (HS), which reflect the proportion of cells positive for TNF-alpha message or protein and the intensities of the signals, were higher for epithelial than for stromal cells. Benign lesions (endometrial polyps) contained little TNF-alpha mRNA or protein, whereas specific message was abundant in proliferative lesions (hyperplasia, adenofibroma). Although neoplastic cells in both low and high grade endometrial tumors contained TNF-alpha mRNA, two major differences were observed: HS for TNF-alpha mRNA were significantly less in low grade than in high grade neoplasms, and TNF-alpha message was restricted to the nucleus in low grade adenocarcinoma cells but was abundant in the cytoplasm of high grade tumor cells. In contrast to cells in benign and proliferative lesions, TNF-alpha protein scores in endometrial tumor cells were inversely rather than positively correlated with TNF-alpha mRNA scores. Collectively, the findings in this study are consistent with the postulate that TNF-alpha is useful to endometrial tumor cells and suggest that production may increase as cells diverge from normal.

Adenocarcinoma↗
PubMed1994

Differential responses of phenotypically distinct rat trophoblast cell lines to MHC class I antigen-inducing cytokines.

Phenotypically distinct rat trophoblast cell lines, the Rcho-1 and R8RP.3 cells, were compared for their responses to cytokines known to induce major histocompatibility (MHC) class I antigens, tumour necrosis factor (TNF), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma). Cell enzyme immunosorbent assays and flow cytometry experiments showed that only IFN-gamma could induce RT1 class I antigens on the Rcho-1 cells. Non-adherent cells were slightly less responsive than adherent, giant cell-like Rcho-1 cells. By contrast, RT1 class I antigens on the R8RP.3 cells were induced by both TGF-beta 1 and IFN-gamma. The cytokines also had different effects on mitochondrial enzyme activity in the two lines. TNF and TGF-beta 1 mRNAs were demonstrated in both lines by using Northern blot hybridization. Rcho-1 but not R8RP.3 cells contained two TNF messages (approximately 2.2, 1.9 kb). Steady state levels of transcripts from the TNF gene, and, to a lesser extent, the TGF-beta 1 gene, were increased in cultures of Rcho-1 cells that contained high proportions of giant cells. Thus, phenotypically distinct rat trophoblast cell lines do not respond identically to TNF, TGF-beta 1 or IFN-gamma, transcription of cytokine genes does not prevent the cells from responding to paracrine cytokine signals, and the cells contain novel TNF transcripts that might be important in cell maturation or differentiation.

Animals↗
PubMed1994

Immunologically relevant cells in the uterus.

Macrophages and a special subset of lymphocyte natural killer (NK) cells populate the uteri of cycling humans, mice, and rats. After implantation, major changes take place that have important functional implications. The macrophages and NK cells increase in number, are redistributed into specific uterine compartments, and exhibit markers consistent with cell activation. Activation enhances macrophage and NK cell production of a wide range of pleiotropic, multifunctional polypeptide growth factors, reactive oxygen intermediates, and bioactive lipids. Thus, activated uterine hematopoietic cells are equipped to perform certain immunological and nonimmunological functions within their microenvironments that could have major influences on the course of pregnancy.

Animals↗
PubMed1994

Mouse endometrial tumor necrosis factor-alpha messenger ribonucleic acid and protein: localization and regulation by estradiol and progesterone.

Tumor necrosis factor-alpha (TNF) messenger RNA (mRNA) and protein have been identified in the uteri of cycling rats, mice, and women. In this study, a mouse model was used to investigate the cell-specific expression and regulation of the TNF gene in the endometrium. Uteri from cycling and ovariectomized hormone-reconstituted mice were tested by in situ and Northern blot hybridizations as well as by immunohistochemistry. Cyclic variations were observed in TNF mRNA. Although weak to undetectable during proestrus, estrus, and diestrus-I, TNF mRNA was present in both epithelial and stromal cells on diestrus-II. TNF protein was observed in the endometrium on each day of the estrous cycle, primarily in the epithelial cells. Stromal TNF immunoreactivity was observed only during diestrus-I. Seven days after ovariectomy, TNF mRNA and protein were undetectable in the endometrium. Specific message and protein were restored in both epithelial and stromal cells after administration of 17 beta-estradiol (E2), progesterone (P4), and E2 plus P4. E2 treatment resulted in a biphasic pattern of TNF mRNA expression. mRNA was present in epithelial cells 1 and 6 h posttreatment, was not detectable after 24 h, and then was present in both the epithelium and stroma after 72 h. In contrast, TNF mRNA was detectable at all time points after P4 administration. TNF mRNA was localized to both the epithelium and stroma until 72 h of P4 treatment, when stromal TNF mRNA was no longer detectable. TNF mRNA was also present at all time points examined after the combination E2 and P4 treatment. TNF mRNA was invariably localized to the epithelium; however, stromal mRNA fluctuated over the course of treatment. TNF mRNA was in the stroma 1 and 24 h post-E2 plus P4 treatment, but was not present at the 6 and 72 h points. In general, the pattern of TNF protein matched that of the TNF mRNA, with a few exceptions. Twenty-four hours after E2 treatment, TNF mRNA was not detectable; however, TNF protein was present in the epithelium. The opposite was observed 24 h after E2 plus P4 administration when the epithelium and stroma contained TNF mRNA; however, no protein was observed. In addition, with the exception of 72 h of E2 treatment, stromal TNF protein was not abundant, although TNF mRNA was often detected. In summary, the results of this study indicate that both epithelial and stromal cells contain TNF mRNA and protein and that E2 and P4 exert individual influences on the cell-specific expression of TNF transcripts and protein.

Animals↗
PubMed1994

Expression of HLA class II-associated peptide transporter and proteasome genes in human placentas and trophoblast cell lines.

Expression of HLA class I antigens is closely controlled in the placental trophoblast cells, which interface directly with maternal cells during pregnancy. In this study, the possibility that peptide transporter (TAP-1, TAP-2) or proteasome (LMP7) genes might be involved in regulating antigen expression in these or other cells that comprise placentas was investigated. Analysis by Northern blot hybridization showed that transcripts from all three genes were present in samples of first trimester and term placental RNA. TAP-1 and TAP-2 messages were consistently more abundant in early than in late gestation placentas, whereas the reverse was observed for LMP7 mRNA. Futher experiments were done on two trophoblast cell lines. One line, Jar, is negative for HLA class I, and the second, JEG-3, expresses HLA-G as well as other HLA class I genes. Both Jar and JEG-3 cells contained TAP-1, TAP-2 and LMP7 mRNA. With the exception of LMP7 in JEG-3 cells, message from all three genes was increased by treating the trophoblast cells with interferon-gamma. While no evidence was collected to support the postulate that the HLA class I negative status of some trophoblast cell subpopulations could be related to absent or dysfunctional TAP-1, TAP-2 or LMP7 mRNA, the data are consistent with the postulate that placental cell expression of HLA class I antigens could be influenced by the availability of peptide transporters and proteasome components.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Cellular distribution of HLA-G mRNA in transgenic mouse placentas.

The human MHC class I gene, HLA-G, is unique among members of the class I gene family in that it is nonpolymorphic, and expression is primarily restricted to extraembryonic tissues. To examine regulatory elements that direct tissue- and cell lineage-specific expression of this gene, transgenic mice expressing HLA-G have been established. In this study, in situ hybridization was used to evaluate the cellular distribution of HLA-G mRNA in transgenic placentas. Extraembryonic tissues were obtained at gestation day 12.5 from embryos that had been microinjected with either 6.0 or 5.7 kb of HLA-G genomic DNA and had been transferred into pseudopregnant HLA-G transgenic mice or Swiss mice. The 6.0 kb transgene contained an additional 250 bp at the extreme 5'-end of the upstream region. Genotype of the recipient had no discernable effect on the cellular distribution of HLA-G mRNA. HLA-G mRNA was present in both trophoblast and mesenchymal cells in transgenic placentas carrying 6.0 kb of genomic HLA-G, a pattern strikingly similar to that of HLA-G message distribution in early gestation human placentas. By contrast, in placentas from embryos carrying 5.7 kb HLA-G DNA, specific mRNA was found primarily in mesenchymal cells at the base of the placenta. Thus, the 6.0 genomic fragment contains elements capable of directing HLA-G expression in placentas, and is particularly influential in the trophoblastic cell lineage.

Animals↗
PubMed1993

Molecular, biochemical, and functional characteristics of tumor necrosis factor-alpha produced by human placental cytotrophoblastic cells.

Previous studies have shown that the TNF-alpha gene is transcribed and translated in fully differentiated human placental syncytiotrophoblast. In this study, TNF transcripts were identified by in situ hybridization in cytotrophoblastic cells, a progenitor subpopulation that proliferates rapidly in early gestation tissues. To establish molecular and biochemical characteristics of cytotrophoblastic TNF and to evaluate potential utilization, experiments were conducted on two cytotrophoblastic cell lines, Jar and JEG-3. Northern blot hybridization and immunocytochemical tests showed that Jar and JEG-3 cells contained TNF mRNA and specific protein. Enzyme immunoassays demonstrated production of TNF, and immunoprecipitation experiments showed that Jar cell TNF protein was the same molecular mass as macrophage TNF. DNA synthesis in both lines was promoted by rTNF, and experiments employing 17-mer TNF antisense and sense oligonucleotides showed specific inhibition of DNA synthesis by antisense sequences. Both p60 and p80 TNF-R mRNA were present in the choriocarcinoma cell lines, and DNA synthesis was inhibited by antibody to the p60 TNF-R. Although the two lines were similar in many respects, Jar cells produced more TNF and demonstrated a greater reliance on TNF for their growth. Collectively, the results indicate that: 1) the TNF gene is expressed in both normal and malignant cytotrophoblast; 2) certain molecular, immunologic, and biochemical characteristics of trophoblast-derived TNF are similar to macrophage TNF; and 3) the p60 TNF-R facilitates utilization of TNF as an autocrine growth factor by choriocarcinoma cells. Although TNF apparently serves important functions in cytotrophoblast during the course of placental development that might include promotion of proliferation and invasion, constitutive expression of this gene in neoplastic cells could account in part for the remarkable ability of trophoblastic tumors to overcome host defenses.

Base Sequence↗
PubMed1993

Immunological studies of lactoferrin in human placentae.

Lactoferrin (LF) and transferrin (Trf) are glycoproteins with strong affinities for ferric ions. Human syncytiotrophoblastic membranes analyzed by enzyme linked immunosorbent assay (ELISA) and immunoblotting were negative with monoclonal and polyclonal antibodies to LF. Immunohistological studies of 35 normal placentae showed that LF was absent from the trophoblast basement membranes, stroma and fetal stem vessel endothelium, but positive cells were occasionally noted in intervillous spaces and fetal stem vessels. In contrast, many LF-positive cells were identified within areas of immunopathology identified by the presence of T cells, HLA-DR-positive macrophages and platelets. Double-antibody experiments showed that the LF-positive cells in these areas reacted with CD15 and CD16 monoclonal antibodies (mAbs), indicating that the cells were polymorphonuclear neutrophils (PMN). PMN from peripheral blood analyzed by flow cytometry and immunocytology also showed reactivities with anti-LF, CD15 and CD16 and we consistently found that circulating PMN reacted better than placental PMN with antibodies to MHC class I antigens and gp 100, (CD67), which is a neutrophil activation marker. PMN adherent within placentae had no detectable MHC class I or CD67 antigens. These findings suggest PMN adherent to placental tissues down-regulate or alter plasma membrane markers. LF appears to play a role in placental inflammation, for LF-positive cells were significantly enriched in areas of immunopathology.

Antibodies, Monoclonal↗
PubMed1993

Identification of transforming growth factor-beta 1 mRNA in virgin and pregnant rat uteri by in situ hybridization.

Transforming growth factor-beta 1 (TGF-beta 1) has major effects on hematopoietic cell proliferation, migration and function. In an effort to learn whether or not TGF-beta 1 might be among the cytokines that influence uterine, placental and embryonic hematopoietic cells, in situ hybridization was used to map expression of the TGF-beta 1 gene through gestation in the rat. In cycling uteri, hybridization signals with a biotinylated TGF-beta 1 antisense RNA probe were comparatively weak and were restricted to myometrium. During pregnancy, TGF-beta 1 mRNA appeared in specific cell lineages in an ordered temporal sequence. In early post-implantation tissues (g.d. 5-8), TGF-beta 1 transcripts were prominent in uterine epithelial cells, muscle and decidual cells. At g.d. 15, hybridization signals were particularly strong in uterine epithelial cells, muscle and placental trophoblast cells. At this stage, TGF-beta 1 mRNA was also present in uterine cells resembling macrophages and in natural killer-like (GMG) cells. Steady state levels of specific message in uteri and placentas declined at late stages of gestation (g.d. 18, 21). In rat embryos, specific transcripts were identified at g.d. 9 and were prominently displayed in various types of embryonic cells from mid-gestation onwards. The results of this study are consistent with the postulate that throughout pregnancy in the rat, TGF-beta 1 may, in addition to its other functions, influence the proportions, patterns of distribution and activities of maternal and fetal hematopoietic cells.

Animals↗
PubMed1993

Expression and regulation of the tumour necrosis factor-alpha gene in the female reproductive tract.

Tumour necrosis factor-alpha (TNF), a potent cytokine originally identified as a product of activated macrophages, is now known to be produced by many types of cells, and has been implicated in regulation of normal tissue homeostasis as well as in cellular differentiation. In humans and some murids, the TNF gene is expressed in ovaries, oviducts, uteri, placentas and embryos. Specific transcripts and proteins have been identified in oocytes, granulosa and theca cells, luminal and glandular epithelial cells, myometrial cells, decidual cells, placental trophoblast, macrophage-like cells and embryonic skin. Both prior and subsequent to implantation, uterine TNF mRNA and protein appear in specific cell lineages in an ordered temporal sequence. This and other findings indicate that transcription of the TNF gene in the uterus is regulated either directly or indirectly by ovarian and/or placental hormones. By contrast, there is as yet no evidence for regulation of this gene by other uterine cytokines such as colony stimulating factor-1 (CSF-1). Although the functions of this pleiotrophic, multifunctional molecule are largely unknown, the findings to date are consistent with the postulate that TNF is involved in gamete development, cyclic changes in the uterus, cancers of the female reproductive tract, placental maturation and embryonic development.

Animals↗
PubMed1993

Tumor necrosis factor-alpha gene expression in mouse oocytes and follicular cells.

Ovarian cells that transcribe and translate the gene for tumor necrosis factor alpha (TNF alpha) were identified in the adult cyclic mouse by using in situ hybridization and immunocytochemistry. TNF alpha mRNA was observed in > 97% and protein was contained in approximately 53% of the oocytes of healthy follicles with two or more layers of granulosa cells, but neither was detectable in oocytes of primordial follicles and follicles with a single layer of granulosa cells. In early atretic follicles, only 13% contained TNF alpha protein and 40% contained TNF alpha mRNA. In late stages of atresia, intense immunoreactive TNF alpha was observed in all of the oocytes, but TNF alpha mRNA was present in only 13%. In approximately 85% of follicles, theca and/or granulosa cells exhibited TNF alpha mRNA hybridization signals. Macrophage-like cells within the interstitium were positive for TNF alpha mRNA and protein. In corpora lutea, luteal cells and macrophage-like cells contained TNF alpha message, while only the latter lineage contained immunoreactive TNF alpha. Hybridization signals and immunoreactivity were more intense in older corpora lutea than in corpora lutea of the present cycle. Northern blot analysis revealed a 2.2-kb TNF alpha mRNA in the ovary that was unchanged relative to 28S rRNA (constitutive RNA) during the cycle. Similarly, TNF alpha hybridization signals and immunoreactivity did not appear to change throughout the cycle. These results indicate that TNF alpha gene transcription in the oocyte coincides with the synthesis of immunoreactive TNF alpha and that these complex biochemical processes occur at distinct steps of follicular development in the mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Normal distribution of tumor necrosis factor-alpha messenger ribonucleic acid and protein in the uteri, placentas, and embryos of osteopetrotic (op/op) mice lacking colony-stimulating factor-1.

In order to map mouse tumor necrosis factor-alpha (TNF) gene expression in detail and to determine whether transcription or translation of the TNF gene is regulated by uterine colony stimulating factor-1 (CSF-1), preimplantation embryos, oviducts, uteri, and uteroplacental units were studied in various strains of mice. These included homozygous osteopetrotic (op/op) female mice, which completely lack CSF-1, and heterozygous (+/op) females, which have normal levels of CSF-1. TNF mRNA was identified in all samples except preimplantation embryos by use of Northern blot hybridization or reverse transcriptase polymerase chain reaction. In situ hybridization and immunocytochemical experiments showed that the TNF gene was expressed in mouse oviduct and uterine epithelial cells, decidual cells, macrophage-like cells, placental trophoblast, and embryos. Despite an absence of CSF-1, TNF gene expression in the uteri, placentas, and embryos of op/op mothers did not differ in any major respect from expression in +/op or other strains of mice. The results of this study therefore indicate that the TNF gene is transcribed and translated in an ordered sequence through mouse gestation, and that maternal CSF-1 is not essential to expression of this cytokine gene. Collectively, these findings are consistent with a major role for TNF in mouse reproduction and development and with a potential compensatory function for this potent polypeptide factor in CSF-1 deficiency.

Animals↗
PubMed1993

Expression of tumor necrosis factor-alpha in mouse spermatogenic cells.

Enriched fractions of spermatogenic cells were isolated by unit gravity sedimentation and analyzed both for the presence of secreted tumor necrosis factor-alpha (TNF alpha) in vitro by bioassay and for the presence of TNF alpha mRNA by Northern blot analysis. Small quantities of bioactive TNF alpha were consistently detected in medium conditioned by round spermatid fractions. Both pachytene spermatocyte and round spermatid fractions contained RNA that hybridized with murine cDNA probes for TNF alpha, with pachytene spermatocytes containing a normal 1.9-kilobase (kb) transcript, while round spermatids contained principally an approximately 2.8-kb transcript. Both the normal size transcript and the larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed through an oligo(dT) column. The normal 1.9-kb transcript within pachytene spermatocytes could be induced by exposing the spermatogenic cells to lipopolysaccharides in vitro, yet the approximately 2.8-kb transcript within round spermatids appeared uninduced by LPS treatment. In situ hybridization for the TNF alpha message by using digoxigenin label antisense TNF alpha riboprobe labeled pachytene spermatocytes, round spermatids, and presumptive interstitial macrophages. Spermatogonia and elongating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis resulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes or round spermatids, hybridized with human TNF alpha receptor p60 probe in Northern blot analysis. These results are consistent with the working hypothesis that spermatids release TNF alpha, which is detected by Sertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.

Animals↗
PubMed1993

Analysis of p60 and p80 tumor necrosis factor-alpha receptor messenger RNA and protein in human placentas.

Tumor necrosis factor-alpha (TNF), a pleiotrophic, multifunctional polypeptide factor, has been reported in both normal and infected human placentas. To identify potential targets for this cytokine, the cells in early and late gestation placentas and extraplacental membranes that express the two TNF receptor (TNF-R) genes, p60 and p80, were identified by using in situ hybridization and immunocytochemistry. Gestation-related, cell lineage-specific differences in steady-state levels of p60 and p80 TNF-R messenger RNA were observed. p60 TNF-R messenger RNA predominated at both early and late stages of gestation, being high in both mesenchymal and trophoblastic cell lineages. By contrast, p80 TNF-R messenger RNA was abundant only in intermittent stretches of first trimester syncytiotrophoblast and term placental mesenchymal cells. Overall, intensities of the TNF-R hybridization signals were stronger in term than in first trimester tissues. Transcription of the two TNF-R genes was confirmed by Northern blot hybridization. Translation was verified in all samples by immunohistology using polyclonal antibodies specific for the receptor proteins. p60 and p80 TNF-R proteins were identified both intracellularly and in maternal and fetal blood. Because TNF-Rs exist in both membrane-bound and soluble forms, the results of this study are consistent with the postulate that placental TNF-R have two critical functions: 1) modulation of TNF utilization by specific placental cell lineages during the course of pregnancy; and 2) protection against excessive TNF produced during infections.

Blotting, Northern↗
PubMed1992

Characterization and use of monoclonal and polyclonal antibodies against the mouse interferon-gamma receptor.

To facilitate investigation of its physical and functional properties, 11 monoclonal antibodies (mAbs) and a goat polyclonal IgG specific for the mouse interferon- (IFN-gamma) receptor were characterized and their potential uses studied. Eight of the mAbs interacted with epitopes on the extracellular domain of the receptor, two interacted with epitopes on the intracellular domain, and one interacted with an epitope that could not be localized definitively to either region. Of the 11 mAbs, the majority (8) were IgGs, 2 were IgMs, and 1 was an IgA. Relative avidities of the seven that could be determined ranged from 333 to 0.002 microM-1. Both the polyclonal goat IgG and mAb GR-20 (the latter specific for an epitope in the binding site for IFN-gamma) blocked binding of the ligand and, as expected, prevented induction by IFN-gamma of priming of macrophages for tumor cell killing. None of the other mAbs had an effect despite the fact that GR-22 partially (greater than 50%) blocked binding of IFN-gamma. Neither the polyclonal IgG nor any of the mAbs had an agonist effect. The relative usefulness of the antibodies for immunoprecipitation, immunoblotting, immunoassay, and cell staining with and without prior fixation is described. The results of immunocytochemical staining directly confirmed that the majority of immunologically reactive receptor protein expressed by cells is intracellular. To facilitate use by other investigators, the hybridomas that produce these mAbs will be offered to the American Type Culture Collection.

Animals↗