Sequence, linkage to H2-K, and function of mouse tapasin in MHC class I assembly.

Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products.

Modulation of macrophage Fc gamma receptors by rGM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor being used increasingly to support white blood cell counts in hematologic disorders. Since the survival of IgG-sensitized cells following blood transfusions and the clearance of immune complexes are important in these disorders, we investigated the effect of GM-CSF on the Fc gamma receptors largely responsible for this immune clearance. Human monocytes were cultured in buffer or 100 U/mL of recombinant GM-CSF (rGM-CSF) for 48 hours. Flow cytometry was used to evaluate changes in the expression of the three Fc gamma receptors. Fc gamma RII was the only Fc gamma receptor significantly increased by rGM-CSF. This increase in Fc gamma RII surface protein was correlated with an increase in macrophage binding of erythrocytes sensitized with IgG. In addition, an increase in monocyte binding of IgG-sensitized RBCs was observed in RBCs sensitized with murine IgG2b antibody, which preferentially binds to Fc gamma RII. rGM-CSF also increased the monocyte Fc gamma RII-dependent low-affinity binding site for trimeric IgG. Furthermore, rGM-CSF was observed to increase the expression of monocyte Fc gamma RII mRNA, including that for Fc gamma RIIA. Thus, these studies demonstrate that GM-CSF increases monocyte Fc gamma RII expression and function and suggests that a similar process may be present in vivo. This effect may be either beneficial (increased clearance of immune complexes) and/or detrimental (increased transfusion requirements) in select patients.

Modulation of the transcriptional rate of Fc gamma receptor mRNA in human mononuclear phagocytes.

Human monocytes and macrophages bear three classes of cell surface receptors for the Fc portion of IgG (Fc gamma RI, Fc gamma RII, and Fc gamma RIII). These receptors mediate phagocytosis and other effector functions and are important in the pathophysiology of hematologic disease, inflammation, and host defense. We have previously shown that interferon-gamma (IFN-gamma) and dexamethasone modulate total Fc gamma RII mRNA levels as well as Fc gamma RI and Fc gamma RII protein expression on monocytes and the monocyte-like cell line U937. In this study, we investigated the modulation of Fc gamma RI mRNA. Additionally, we utilized mRNA stability and nuclear run-on assays to study the mechanism of the modulation of Fc gamma receptor transcripts in the monocyte/macrophage cell line U937. In U937 cells, IFN-gamma increased Fc gamma RI mRNA levels 7.5-fold. Treatment with dexamethasone reduced Fc gamma RI mRNA levels to 0.6-fold of baseline and inhibited by 20-60% the increase in mRNA observed after treatment of the U937 cells with IFN-gamma. On monocytes, treatment with IFN-gamma increased monocyte Fc gamma RI mRNA 6.7-fold. Cotreatment of the IFN-gamma-stimulated monocytes with dexamethasone resulted in a 160% further increase in Fc gamma RI expression. Fc gamma RI and Fc gamma RII mRNA half-lives were then determined in U937 cells by incubation with dexamethasone and/or IFN-gamma for 16 hr and then arresting mRNA transcription with actinomycin-D (10 micrograms/ml). The mRNA half-lives in untreated U937 cells were 3.3 +/- 0.3 hr (Fc gamma RI) and 3.1 +/- 0.3 hr (Fc gamma RII). For either Fc gamma RI and Fc gamma RII, the effect of dexamethasone and/or IFN-gamma on mRNA half-life was not significant (P > 0.5). We also performed nuclear run-on experiments with U937 cells which indicated that IFN-gamma increased the transcription of Fc gamma RI 4.2-fold and Fc gamma RII 1.7-fold. Our data suggest that these changes in Fc gamma RI and Fc gamma RII protein are likely due, at least in part, to increases in mRNA levels secondary to alteration in gene transcription.

Characterization of Fc gamma receptors on a human erythroleukemia cell line (HEL).

It has been established that human platelets express a single class of Fc gamma receptors that has been designated Fc gamma RII. However, the function of this receptor on these cells and its regulation are less certain. Studies to further investigate Fc gamma RII on platelets are limited by the inability to culture platelets in vitro. Therefore, identification of a human cell line that expresses Fc gamma RII as its only Fc gamma receptor as well as other platelet characteristics would be of potential importance. To this end, we examined Fc gamma receptor expression by the human erythroleukemia (HEL) cell line, which expresses platelet/megakaryocyte surface proteins. Flow cytometry studies on HEL cells with anti-Fc gamma receptor monoclonal antibodies revealed that, similar to platelets and megakaryocytes, Fc gamma RII is the only Fc gamma receptor expressed on the cell surface. Furthermore, Northern blot analysis revealed that Fc gamma RII is the only Fc gamma receptor mRNA present. Stimulation with dimethylsulfoxide (DMSO) or 12-O-tetradecanoylphorbol-13-acetate (TPA) did not alter Fc gamma RII protein or mRNA expression. Ligand binding studies with [125I]IgG trimer indicated that HEL cells express 92,240 +/- 5030 binding sites per cell, with a kd of 1.94 +/- 0.31 x 10(-8) M. Similar to human platelets, HEL cells preferentially bound oligomeric IgG, and this binding was ionic strength dependent. These observations are similar to those previously observed with Fc gamma RII on human platelets and suggest that the HEL cell Fc gamma receptor is similar, if not identical to the platelet Fc gamma RII receptor. HEL cells may serve as a model for the study of platelet/megakaryocyte Fc gamma RII.

cDNA cloning and characterization of interleukin 2-induced genes in a cloned T helper lymphocyte.

Clonal expansion of antigen-specific lymphocytes is an important aspect of the immune response. Interleukin 2 (IL2) is largely responsible for the amplification of antigen-specific T cells. In this study, the changes in gene expression accompanying interleukin 2 stimulation of T cells are examined, using a cloned T helper lymphocyte line as a model system. To isolate cDNA clones of IL2-induced genes, a cDNA library was screened by differential hybridization. Twenty-one different cDNA clones were isolated by this method, comprising six glycolytic enzymes, vimentin, alpha-tubulin, beta-actin, gamma-actin, ERp99, elongation factor 2, ribosomal phosphoprotein P1, the DNA-binding protein dbpB/YB-1, as well as seven clones which do not correspond to any previously described sequences. These clones are used to study the time course of expression and the sensitivity to cycloheximide inhibition of IL2-induced mRNAs. In addition, the tissue specificity of the unidentified mRNAs is examined, and two of these are shown to be expressed at high levels in normal mouse brain, with much lower or undetectable levels in the other tissues tested. These cDNA clones will be useful in future studies to determine the molecular basis of IL2-induced gene expression.

Modulation of human mononuclear phagocyte Fc gamma RII mRNA and protein.

Human monocytes and macrophages express three different classes of cell surface receptors for the Fc portion of IgG, Fc gamma RI (CD64), Gc gamma RII (CD32), and Fc gamma RIII (CD16). We utilized a cDNA probe for Fc gamma RII to examine the modulation of Fc gamma RII mRNA by dexamethasone, a synthetic glucocorticoid, and interferon-gamma. We also determined the changes in the expression of both Fc gamma RI and Fc gamma RII protein following treatment with these agents by flow cytometry. In studies performed with the monocyte-like cell line. U937, Northern blot analysis revealed that cells treated with interferon-gamma showed a 2.5-fold increase in Fc gamma RII mRNA levels that was maximal at 14 hr and declined to 1.4-fold over baseline by 48 hr of incubation. Treatment of U937 cells with dexamethasone did not significantly change the level of Fc gamma RII transcripts, but was able to inhibit by up to 50% the increase seen following interferon-gamma treatment. The expression of Fc gamma RII protein on U937 cells was increased 56-72% after 16-24 hr of interferon-gamma treatment, but was only 18% over baseline after 48 hr of incubation. Treatment with dexamethasone caused a small, but significant, decrease in Fc gamma RII protein, and inhibited by 20-60% the induction of Fc gamma RII by interferon-gamma. The modulation by dexamethasone and interferon-gamma of Fc gamma RI protein expression on U937 cells was markedly different from that of Fc gamma RII in both magnitude and kinetics. Interferon-gamma treatment increased Fc gamma RI expression by 240% at 16 hr, and Fc gamma RI remained elevated through 48 hr. Treatment with dexamethasone decreased Fc gamma RI expression by 39%, and also inhibited by 40% the increase induced by interferon-gamma. In contrast to the findings with U937 cells, dexamethasone and/or interferon-gamma treatment had no significant effect on Fc gamma RII mRNA levels or protein expression in monocytes. However, interferon-gamma treatment increased Fc gamma RI expression on monocytes, and this increase was further augmented by treatment with dexamethasone. These data indicate that the modulation of Fc gamma RII on U937 cells is at least in part due to changes in steady state levels of Fc gamma RII mRNA. The difference between the magnitude of the changes in Fc gamma RII mRNA and protein suggests that some translational or post-translational control is involved in regulating the expression of Fc gamma RII.(ABSTRACT TRUNCATED AT 400 WORDS)

Receptors for the Fc portion of immunoglobulin G (Fc gamma R) on human monocytes and macrophages.

Human mononuclear phagocytes bear a group of cell surface receptors that bind the Fc domain of IgG. These Fc gamma receptors are utilized in the phagocytosis of opsonized cells and immune complexes and are involved in the pathogenesis of several hematologic and immunologic disorders. However, the relative contributions of the different Fc gamma receptors to these processes is uncertain. The expression of these receptors can also be modulated by several physiologic and pharmacologic factors, including IFN-gamma and glucocorticoids, but the mechanisms of this modulation have not been fully elucidated. The recent isolation of molecular probes for these receptors should allow a more complete understanding of the function of these molecules and their response to modulatory signals.

Cyclin mRNA and protein expression in recombinant interleukin 2-stimulated cloned murine T lymphocytes.

Expression of cyclin, a non-histone nuclear protein, during recombinant interleukin 2 (rIL2)-driven cell-cycle progression of cloned T lymphocytes has been assessed. We found that expression of cyclin protein, as detected by immunofluorescence, is tightly associated with proliferation, and not merely S-phase, of L2 cells stimulated with rIL2. Cyclin immunofluorescence was detected in all cell-cycle phases (G1/S/G2/M, as detected by flow cytometry) of proliferating L2 cells. Accumulation of cyclin mRNA levels was induced as early as 1 h after stimulation, was maximal at 25-49 h, and remained elevated throughout stimulation, as detected by Northern blot analysis. A cDNA-encoding murine cyclin was cloned from a cDNA library prepared from IL2-stimulated cloned T cells. The sequence of the 5' end of the murine cyclin cDNA was determined and found to be 88% and 82% similar to the sequences of cDNA clones encoding rat and human cyclin, respectively. The present studies demonstrate that cyclin protein and mRNA accumulation are highly regulated during IL2-induced proliferation of a cloned T cell. These data provide a framework for addressing the molecular mechanisms regulating cyclin gene expression during cellular proliferation.