IFN-gamma Induction Research Articles

Cytokine induction in HTLV‐I associated myelopathy and adult T‐cell leukemia: Alternate molecular mechanisms underlying retroviral pathogenesis

The human T-cell lymphotropic virus type I (HTLV-I) is capable of inducing a variety of host cellular genes including many of the cytokines responsible for immune regulation and osteoclast activation. This derangement in cytokine expression may contribute to the panoply of disease states associated with HTLV-I infection such as the adult T-cell leukemia (ATL) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). We wished to determine if there was a correlation between the expression of an array of cytokines and the diverse clinical manifestations of ATL and HAM/TSP. Utilizing the techniques of specific mRNA amplification by the polymerase chain reaction (PCR) as well as Northern blotting, we analyzed the ex vivo mRNA expression of gamma-interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and transforming growth factor-beta 1 (TGF-beta 1) in the peripheral blood of HAM/TSP and ATL patients as well as asymptomatic seropositive carriers. IFN-gamma, TNF-alpha, and IL-1 beta transcripts were up-regulated in patients with HAM/TSP and seropositive carriers when compared to their levels in ATL and normal controls. In contrast, the ATL patients constitutively expressed higher levels of TGF-beta 1 mRNA than HAM/TSP and seropositive carriers. In addition, TNF-alpha and IL-1 beta serum levels were elevated in HAM/TSP, but not in ATL patients nor seropositive carriers. However, the circulating leukemic cells from ATL patients secreted increased levels of TGF-beta 1 protein into the culture medium than T-cells derived from HAM/TSP patients. Collectively these results suggest that induction of IFN-gamma, TNF-alpha, and IL-1 beta in HAM/TSP may initiate an inflammatory cascade with subsequent events leading to immune mediated destruction of the central nervous system in these patients. Expression of osteoclast activators such as TNF-alpha and IL-1 beta is not associated with hypercalcemia in ATL. Finally, impaired cellular and humoral immune responses present in ATL, but not in HAM/TSP, may be related to elevated levels of TGF-beta 1 produced by the leukemic cells. These differences in retroviral-induced host cytokine expression in ATL and HAM/TSP suggest alternate roles in disease pathogenesis.

Immunomodulation of human cytomegalovirus infection on interferon system in patients with systemic lupus erythematosus

On the basis of the research on the relationship between human cytomegalovirus (HCMV) infection and systemic lupus erythematosus (SLE) a study of the effects of HCMV infection on the immunological pathogenesis of SLE was undertaken. Presented in this paper is a brief account of the study. It was found that SLE patients with active HCMV infection had significant higher level of serum interferon-alpha (IFN-alpha) than those without active HCMV infection, but the IFN-alpha induction capacity of peripheral blood mononuclear cells (PBMC) from SLE patients with active HCMV infection was evidently poorer than that from patients without it. There was not any statistically significant difference between the two SLE groups in the IFN-gamma induction capacity of PBMC.

Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.

Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.

Cyclic AMP synergy with Ca2+ for production of IFN-gamma by a cytolytic T cell clone is post-transcriptional.

PGE2 or products increasing the intracellular concentration of cAMP (cAMP)i) had opposite effects on the induction of IFN-gamma in a CTL clone, depending on the inducing agent. Activation via the TCR was inhibited, whereas induction by the Ca2+ ionophore ionomycin was enhanced in the presence of agents increasing (cAMP)i. Synergy between Ca2(+)-dependent and cAMP-dependent pathways was independent of the activation of protein kinase C (PKC). Low levels of IFN-gamma mRNA could be detected transiently after induction with ionomycin alone, whereas simultaneous induction with agents increasing (cAMP)i led to enhanced levels of IFN-gamma mRNA detectable up to 12 h. No IFN-gamma mRNA was detected when the CTL were activated with (cAMP)i-increasing agents alone or with PKC-activating agents such as PMA, suggesting that the transcriptional activation of the IFN-gamma gene was strictly dependent on the Ca2(+)-mediated and cyclosporin A-dependent event. Analyses of IFN-gamma mRNA transcription by "run-on" experiments on nuclei isolated after activation of the CTL indicated that the Ca2+ signal alone induces maximal transcription of the IFN-gamma gene, which is not increased by either PKC activation or an increase in cAMP, but that further processing or stabilization of the IFN-gamma precursor or mature mRNA require an additional signal, provided either via a PKC or via a PKA activation pathway. The data also suggest that a combination of inflammatory products leading to an increase in (Ca2+)i and to an increase in (cAMP)i may bypass the usually stringent control of T cell activation by the TCR/CD3 complex.

Amiloride does not influence the capability of interferon gamma to potentiate superoxide anion and hydrogen peroxide release by human mononuclear phagocytes.

Since the molecular mechanisms of macrophage activation in response to interferon gamma (IFN-gamma) are still not well defined we have investigated whether amiloride, a specific inhibitor of the Na+/H+ antiporter, had any effect on the IFN-gamma-mediated potentiation of human monocyte and monocyte-derived macrophage capability to produce O2- (respiratory burst). Here, we demonstrate that amiloride neither inhibits the capability of IFN-gamma to activate the mononuclear phagocyte respiratory burst nor influences IFN-gamma induction of steady-state mRNA levels for 2 components of the superoxide anion-generating enzyme system. On the contrary, we show that IFN-gamma-enhanced expression of the HLA-DR alpha gene is significantly inhibited by amiloride These data indicate that Na+/H+ antiporter stimulation by IFN-gamma is not involved in the mechanism of activation of macrophage oxidative metabolism.

Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers.

We previously reported that natural killer cell stimulatory factor (NKSF), a heterodimeric lymphokine purified from the conditioned medium of human B lymphoblastoid cell lines, induces interferon gamma (IFN-gamma) production from resting peripheral blood lymphocytes (PBL) and synergizes with interleukin 2 in this activity. In this study, we show that human NKSF induces IFN-gamma production from both resting and activated human PBL and from freshly isolated murine splenocytes. Human T and NK cells produce IFN-gamma in response to NKSF, but resting PBL require the presence of nonadherent human histocompatibility leukocyte antigens DR+ (HLA-DR+) accessory cells to respond to NKSF. The mechanism(s) by which NKSF induces IFN-gamma production results in accumulation of IFN-gamma mRNA, is insensitive to cyclosporin A, and synergizes with those mediated by phytohemagglutinin, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens, but not by Ca2+ ionophores. The ability of NKSF to directly induce IFN-gamma production and to synergize with other physiological IFN-gamma inducers, joined with the previously described ability to enhance lymphocyte cytotoxicity and proliferation, indicates that this lymphokine is a powerful immunopotentiating agent.

5' regulatory region of a novel cytokine gene mediates selective activation by interferon gamma.

A newly described member of the platelet factor 4 family of cytokine genes, mig, is selectively induced by interferon gamma (IFN-gamma), and not IFN-alpha, in the mouse macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with IFN-gamma activated mig gene transcription as determined by nuclear run-on assays. mig genomic clones were isolated, and constructs containing genomic fragments that included the mig promoter region and the CAT reporter gene were prepared. In RAW 264.7 cells transfected with these constructs, CAT activity was found to be selectively induced by IFN-gamma. A 278-bp genomic fragment containing 235 nucleotides 5' of the transcription start site was sufficient for IFN-gamma-selective induction of CAT activity. Analysis of 5' deletion mutants localized a region essential for activation by IFN-gamma to within 64 nucleotides extending from -235 to -172. A genomic fragment containing this sequence was capable of conferring IFN-gamma inducibility to constructs with a heterologous promoter.

Inducibility of class II major histocompatibility complex antigens by interferon gamma is associated with reduced tumorigenicity in C3H mouse fibroblasts transformed by v-Ki-ras.

Paired lines of C3H mouse fibroblasts transformed with murine sarcoma virus (Kirsten strain) were prepared that express high or low levels of class II major histocompatibility complex antigen after treatment with interferon gamma (IFN-gamma). Here, we described a comparison of the tumorigenicity of these lines in euthymic syngeneic and thymus-deficient nu/nu mice and in mice depleted of IFN-gamma. The class II-inducible cells are clearly less tumorigenic than the noninducible cells in syngeneic mice, but of similar tumorigenicity in nu/nu mice and in mice treated with antibodies to deplete IFN-gamma. We propose that in this system, IFN-gamma induction of class II antigens on the tumor cell surface operates to limit tumor growth; ras expression, which inhibits induction of class II antigens, prevents this and so allows tumor growth.

Isolation and characterization of the promoter region of the human intercellular adhesion molecule-1 gene

We have isolated and restriction enzyme-mapped a human genomic DNA clone encompassing the first two exons and the 5' flanking sequence of the human intercellular adhesion molecule-1 (ICAM-1) gene. The transcription initiation site was identified using primer extension analysis, and 1.7 kb of DNA upstream of the transcription initiation site was sequenced. The 5' region and first exon of the ICAM-1 gene was found to be a CpG island as it was (i) (G + C)-rich with a high frequency of the dinucleotide CpG and (ii) hypomethylated irrespective of the level of ICAM-1 expression in the tissues examined. These features of the ICAM-1 promoter are similar to the promoters of many 'housekeeping' genes. However, consensus sequences for several potential regulatory elements were found in the 5'-flanking sequence, an observation in keeping with the pattern of strongly regulated ICAM-1 expression. Examination of the chromatin structure upstream of the ICAM-1 gene revealed the presence of a constitutive DNase I-hypersensitive site 1.5 kb upstream of the transcription initiation site. Direct evidence that the upstream region constitutes a promoter element was demonstrated in transient transfection assays. A series of chloramphenicol acetyltransferase gene (CAT) constructs containing 5' fragments ranging in size from 1054 to 310 bp had equivalent levels of promoter activity when transfected into HeLa cells. Using a CAT construct containing a 447 bp ICAM-1 promoter fragment, we demonstrate an increase in transcription in response to interferon gamma (IFN-gamma), suggesting that this proximal region of the promoter is responsible, at least in part, for IFN-gamma induction of ICAM-1 expression.

The effects of blockade of interleukin 2 receptors and interleukin 4 receptors on cytokine production

The understanding of the regulation of cytokine production and T-cell proliferation by other cytokines is far from complete. We have examined the supernatant concentration of different cytokines in cultured peripheral blood lymphocytes (PBL) incubated with monoclonal antibodies against the interleukin-2 receptor (IL-2R) or interleukin-4 receptor (IL-4R) complex in order to analyse the importance of these receptors in the production of other cytokines. The PBL cultures were stimulated with phytohaemagglutinin and phorbol myristate acetate. The addition of anti-Tac to PBL cultures resulted in increased free IL-2 due to blockade of IL-2R. MR6 (a monoclonal antibody against IL-4R) did not affect the supernatant concentration of IL-2 in cultured PBL. Blockade of IL-2R or IL-4R both resulted in increased IL-4 in supernatant. The increase in supernatant concentration of IL-4 in PBL cultured with MR6 was due to a direct interaction with the IL-4R complex but the increase in IL-4 with anti-Tac was due to the accumulation of free IL-4 as an effective blockade of IL-2R prevented the full expression of IL-4R. Neither anti-Tac nor MR6 affected the IL-6 production by activated PBL, suggesting the existence of an IL-2- and IL-4-independent pathway. Blockade of IL-2R or IL-4R both resulted in a reduction of interferon-gamma (IFN-gamma) production, indicating that both endogenously generated IL-2 and IL-4 are important mediators of IFN-gamma induction in PBL cultures stimulated with T-cell mitogens.

Atterns of Lymphokine Production by Primary Antigen-Specific/MHC Restricted Murine Helper T Cell Clones

In this study we examined a panel of CD4+ antigen specific/MHC restricted T cell clones for their ability to secrete IL-2, IL-4, and IFN-gamma upon stimulation with con A, three lymphokines which are diagnostic for the TH1 and TH2 subtypes of helper T cells. Eight of the twelve clones we analyzed did not fit the classical TH1/TH2 patterns of lymphokine secretion. Seven of these clones secreted both IL-2 and IL-4 and two of these also produced IFN-gamma. The remaining non-classical clone secreted IL-4 and IFN-gamma but not IL-2. Data from the subcloning of the IL-2/IL-4/IFN-gamma triple producers were not consistent with the parental lines being a mixture of TH1 and TH2 cells. The IL-2/IL-4 double producers (IFN-gamma negative) cannot be explained by the parental lines being a mixture of the TH1 and TH2 subtypes. Nevertheless, these double producers were subcloned and the results provided convincing evidence that clones which secrete both IL-2 and IL-4 do exist. Lymphokine loss variants involving IL-2, IL-4 or IFN-gamma were observed among subclones derived from the double and triple producers as well as in several parental lines maintained in continuous culture. We also observed the appearance of inducible IFN-gamma production in some subclones derived from parental clones where production of IFN-gamma was not detectable. The phenotypes of these variants failed to indicate an obvious trend toward the TH1 and TH2 subtypes. Thus, our results suggest that more heterogeneity in the population of CD4+ helper T cells exists than can be explained by the TH1 and TH2 subtypes of these cells.

Interferon gamma response region in the promoter of the human DPA gene.

The interferon gamma (IFN-gamma) response region of the human class II major histocompatibility complex gene, DPA, has been localized to a 52-base-pair (bp) DNA fragment in the proximal promotor at -107 to -55 bp after transfection into HeLa cells of a series of 5', 3', and gap deletion mutants linked to a reporter gene, human growth hormone, as well as of synthetic oligonucleotides fused to the heterologous promoter thymidine kinase. The 52-mer sequence contains the X and Y box elements conserved in all class II genes; their presence is indispensable for IFN-gamma inducibility. Furthermore, an additional 5 bp immediately 5' of the X box of the DPA gene are necessary and sufficient for IFN-gamma induction. This region may contain an IFN-gamma response element. A closely related sequence has also been found in the vicinity of the critical deletion sites of three other well-studied class II gene promoters, all of which require a much longer sequence 5' of the X box. A fourth element, the W element, located about 15 bp 5' of the X box in all class II genes, is clearly of little importance in IFN-gamma inducibility of the DPA gene.

Monocytes and polymorphonuclear neutrophils of patients with streptococcal pharyngitis express increased numbers of type I IgG Fc receptors.

Studies using cultured cells have shown that gamma interferon (IFN-gamma) induces the expression of Fc gamma RI (the type I Fc receptor for IgG) on human polymorphonuclear neutrophils (PMN) and greatly increases the number of these receptors on human monocytes. Administration of rIFN-gamma in vivo also causes enhanced Fc gamma RI expression on these cell populations. Because streptococcal antigens are potent inducers of IFN-gamma in vitro, we postulated that IFN-gamma would be produced endogenously in vivo in patients with streptococcal infections. Such production of IFN-gamma in vivo, even at low levels, might be expected to induce the expression of Fc gamma RI on monocytes and neutrophils. To evaluate this possibility, we used monoclonal antibody 32 (mAb 32), which is specific for Fc gamma RI, to quantitate the expression of this receptor on human peripheral blood cells. We measured the binding of mAb 32 to monocytes and PMNs isolated from healthy donors and from patients with group A beta-hemolytic streptococcal (GABHS) pharyngitis. PMNs from healthy donors (n = 12) had 700 +/- 600 (mean +/- SD) mAb 32 binding sites. Patients with pharyngitis and negative throat culture for GABHS (n = 11) had 2,100 +/- 1,600 sites on their PMNs. In contrast, the PMNs from patients with documented GABHS pharyngitis (n = 12) had 11,600 +/- 7,500 mAb 32 binding sites on their surface. There was a similar change in the expression of Fc gamma RI on monocytes, with control monocytes having a mean of 19,900 +/- 3,200 mAb 32 binding sites per cell and the GABHS-positive monocytes having 47,500 +/- 21,400 sites. The GABHS-negative throat culture group had a slightly elevated number of Fc gamma RI with a mean of 28,200 +/- 8,400 sites. 10 patients with documented urinary tract infections and three patients with uncomplicated pyelonephritis had no elevation in Fc gamma RI expression. These studies demonstrate that a localized group A streptococcal infection can cause systemic activation of the entire circulating pool of phagocytes, and suggest that a similar level of activation is uncommon in localized gram-negative infections of the urinary tract.

Lymphocyte-mediated activation of cultured endothelial cells (EC). CD4+ T cells inhibit EC class II MHC expression despite secreting IFN-gamma and increasing EC class I MHC and intercellular adhesion molecule-1 expression.

Endothelial cells (EC) were cocultured with allogeneic PBL, CD4+ T cells, or CD8+ T cells, and the degrees of EC activation induced examined by determining patterns of endothelial class I and class II MHC and intercellular adhesion molecule-1 (ICAM-1) expression. Coculture with PBL or CD8+ T cells uniformly increases class I MHC and ICAM-1 expression on all EC within a culture, but induces class II MHC expression on only a subpopulation(s) of EC. This heterogeneous EC response to coculture contrasts with the uniform class II expression on all EC induced by IFN-gamma in replicate wells. CD4+ T cells, when compared to equal numbers of unfractionated PBL or CD8+ T cells, are more effective at increasing class I MHC and ICAM-1 but are unable to induce class II MHC expression. The failure of CD4+ T cells to induce EC class II MHC Ag is not due to insufficient activation of the T cells, as PHA-activated CD4+ T cells also do not induce significant class II expression. In addition, conditioned media (CM) from CD4+ T cell/EC contain greater levels of immunoreactive IFN-gamma than do CM from PBL/EC cocultures. Rather, CD4+ T cells appear to actively inhibit the induction of EC class II Ag but not class I or ICAM-1 by IFN-gamma. Inhibition occurs at the time of induction, as CD4+ T cells are not capable of down-regulating previously induced class II Ag. CM from CD4+/EC (but not PBL/EC) cocultures also inhibits IFN-gamma induction of EC class II MHC expression. The inhibitory activity is generated during CD4+ T cell-EC cell contact, and is enhanced by PHA. The inhibitory activity(ies) of the CD4+/EC-CM is as yet unidentified, and is only minimally reversible by cocktails of neutralizing antibodies directed against TNF-alpha, TNF-beta (lymphotoxin), IFN-alpha and IFN-beta. In conclusion, CD4+ and CD8+ T cells are each effective activators of EC, but the patterns of activation produced by these subsets are quite distinct, largely due to generation of a soluble inhibitor(s) of class II MHC induction during coculture of CD4+ T cells with EC.

The IFN-gamma response of the murine invariant chain gene is mediated by a complex enhancer that includes several MHC class II consensus elements.

IFN-gamma induces the expression of the MHC class II-associated invariant chain (IN) protein in a variety of cells of nonlymphoid origin. Here we analyze the transcription from the murine invariant chain gene and delimit the cis-acting sequences which confer IFN-gamma responsiveness in human fibroblasts. The major start of transcription of the gene is located 29 bp 3' of a TATA box and 85 bp 5' of the single ATG codon which opens the reading frame. To identify the regulatory elements of the murine IN promoter which respond to IFN-gamma, the 5' flanking region of the gene including its capsite and 85 bp of coding region have been cloned in front of the bacterial chloramphenicol acetyl transferase (CAT) gene. Examination of this construct and various 5' and 3' deletion mutants for IFN-gamma inducibility in transient transfection assays revealed that DNA sequences between -261 and -189 were essential and sufficient for the induction. Removal of sequences between -215 and -189 reduced inducibility of the IN-promoter and abolished the capacity of the element to transmit inducibility to a heterologous promoter. Single or multiple base changes in other parts of the element also abolished inducibility. Cotransfection of a 350 molar excess of the IFN-gamma response element with an inducible IN-CAT chimeric construct blocked inducibility, suggesting positive regulation. A protein binding to the central part of the IFN-gamma response element was detectable in gel retardation experiments; it was active only in extracts from IFN-gamma-treated cells.

1α, 25-Dihydroxyvitamin D 3 modulates Ia antigen expression induced by interferon-γ and prostaglandin E 2 production in Pam 212 cells

Recent investigations suggest that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] has an effect on the regulation of immune responses and that keratinocyte (KC) expression of major histocompatibility complex class II antigens may be involved in immune responses. We investigated the modulation by 1 alpha,25(OH)2D3 of Ia antigen expression induced by interferon-gamma (IFN-gamma) and prostaglandin E2 (PGE2) production in Pam 212 cells. 1 alpha,25(OH)2D3 at 10(-6), 10(-8) or 10(-10) M significantly decreased the levels of IFN-gamma-induced Ia antigen expression in Pam 212 cells. Pam 212 cells produced PGE2 and 1 alpha,25-(OH)2D3 enhanced Pam 212 cell PGE2 production. However, indomethacin (1, 5 and 10 micrograms/ml) did not abrogate the inhibitory effect of 1 alpha,25(OH)2D3 on IFN-gamma induction of Ia antigen expression in Pam 212 cells, indicating that the products of the cyclo-oxygenase pathway do not mediate 1 alpha,25(OH)2D3 inhibition of IFN-gamma induction of Pam 212 cell Ia antigen expression. Our studies suggest that in Pam 212 cells the levels of Ia antigen expression induced by IFN-gamma and PGE2 production are negatively and positively regulated by 1 alpha,25(OH)2D3, respectively, and that 1 alpha,25(OH)2D3 may play a role in the regulation of immune responses.

Mutational Analysis of the DRA Promoter: cis-Acting Sequences and trans-Acting Factors

Class II major histocompatibility genes are expressed at high levels in B lymphocytes and are gamma interferon (IFN-gamma) inducible in many other cells. Previously, we observed that DRA promoter sequences from positions -150 to +31 determine the tissue specificity of this class II gene. Moreover, Z and X boxes located between positions -145 and -87 conferred B-cell specificity and IFN-gamma inducibility upon a heterologous promoter. In this study, sequences from positions -145 to -35 in the DRA promoter were systematically mutated by using oligonucleotide cassettes. Z (-131 to -125), pyrimidine (-116 to -109), X (-108 to -95), Y (-73 to -61), and octamer (-52 to -45) boxes were required for B-cell specificity and, with the exception of the octamer box, for IFN-gamma inducibility. Z box and sequences flanking Z and X boxes helped to determine low levels of expression in T and uninduced cells. In phenotypically distinct cells, shared and distinct proteins bound to these conserved upstream sequences. However, few correlations between expression and DNA-binding proteins could be made. Similar proteins bound to Z and X boxes, and the Z box most likely represents a duplication of the X box.

Mutational analysis of the DRA promoter: cis-acting sequences and trans-acting factors.

Class II major histocompatibility genes are expressed at high levels in B lymphocytes and are gamma interferon (IFN-gamma) inducible in many other cells. Previously, we observed that DRA promoter sequences from positions -150 to +31 determine the tissue specificity of this class II gene. Moreover, Z and X boxes located between positions -145 and -87 conferred B-cell specificity and IFN-gamma inducibility upon a heterologous promoter. In this study, sequences from positions -145 to -35 in the DRA promoter were systematically mutated by using oligonucleotide cassettes. Z (-131 to -125), pyrimidine (-116 to -109), X (-108 to -95), Y (-73 to -61), and octamer (-52 to -45) boxes were required for B-cell specificity and, with the exception of the octamer box, for IFN-gamma inducibility. Z box and sequences flanking Z and X boxes helped to determine low levels of expression in T and uninduced cells. In phenotypically distinct cells, shared and distinct proteins bound to these conserved upstream sequences. However, few correlations between expression and DNA-binding proteins could be made. Similar proteins bound to Z and X boxes, and the Z box most likely represents a duplication of the X box.

Enhancement of Interferon-γ–Induced Differentiation of Human Monoblastic Leukemia U-937 Cells by cAMP-Inducing Agents

The effects of combinations of interferons (IFNs) and cAMP-inducing agents on the induction of differentiation of human monocytic leukemia U-937 cells were examined. IFN-gamma induced nitro blue tetrazolium (NBT) reducing activity of U-937 cells in a dose-dependent manner, while cAMP-inducing agents such as cholera toxin, prostaglandin E1, forskolin, and isoproterenol only marginally induced NBT reducing activity. However, they all synergistically increased IFN-gamma induction of NBT reducing activity. Cholera toxin was the most potent of the cAMP-inducing agents. Combination effects of IFN-gamma and cholera toxin on other differentiation-associated markers of alpha-naphthyl acetate esterase activity, morphological maturation, Fc receptors, and surface phenotype were also observed. IFN-alpha and -beta, either alone or in combination with cAMP-inducing agents, did not induce NBT reducing activity. IFN-gamma and cholera toxin also synergistically induced differentiation-associated markers in another human monocytic leukemia cell line, THP-1, and a human myeloblastic leukemia cell line, ML-1. These results suggest that cAMP/A-kinase may be an important but insufficient signal for the maturation process of myelogenous leukemia cells.

MHC antigen induction by interferon gamma on cultured mouse pancreatic beta cells and macrophages. Genetic analysis of strain differences and discovery of an "occult" class I-like antigen in NOD/Lt mice.

This study provides a basis for understanding the wide variations reported in the literature in IFN-gamma inducibility of class II MHC antigens on murine beta cells. Inducibility is not an intrinsic property of all mouse beta cells, but instead depends upon strain- (and tissue-) specific response modifying factors. This was demonstrated by comparison of constitutive and IFN-gamma-induced class I and class II MHC gene products on cultured islet cell monolayers. Islet cultures were established from autoimmune diabetes-prone NOD/Lt mice, diabetes-resistant NON/Lt and CBA/J mice, as well as F1 hybrids between these latter two strains and NOD/Lt. Cultures of peritoneal macrophages (M phi) from each strain were established as controls. After 3 wk of culture (with incubation in the presence or absence of IFN-gamma during the last 6 d), constitutive expression as well as IFN-gamma induction of class I MHC antigen expression was demonstrated on NOD/Lt and NON/Lt islet cells by antibody plus complement-mediated cytotoxicity. Although CBA/J islets and M phi did not maintain constitutive class I or class II antigen expression in culture in the absence of IFN-gamma, class I H-2Kk antigen was IFN-gamma inducible. Whereas IFN-gamma-induced class II I-Ak antigen on CBA/J M phi, it failed to induce this antigen on CBA/J islets. In contrast, I-A antigens were IFN-gamma inducible on NOD/Lt and NON/Lt islets and M phi. In (CBA x NOD)F1 hybrids, loss of IFN-gamma inducibility of the I-ANOD product established that suppression was mediated by a trans-acting factor from the CBA/J genome. In the course of these studies, IFN-gamma inducibility of a crossreactive occult class I-like antigen on both NOD/Lt islet cell and M phi cultures was unexpectedly detected when mAb 28-13-3 (public specificity 39, reactive with H-2Kb,f) was used as a negative control. Although not detectable by cytofluorographic analysis of freshly isolated NOD/Lt splenic leukocytes, occult antigen could be induced on NOD/Lt peritoneal macrophages (M phi) cultured for 3 d in IFN-gamma. Time course of induction showed the occult antigen to be distinct from NOD/Lt class I and II gene products. In both islet cell and M phi cultures established from (CBA x NOD)F1 hybrids, trans-suppressive factor(s) from the CBA/J genome not only suppressed IFN-gamma-induced expression of I-ANOD, but additionally suppressed occult antigen induction. Backcross of F1 to both parental strains indicated that the occult locus was on Chr 17, tightly linked to MHC.(ABSTRACT TRUNCATED AT 400 WORDS)