Induction Of Ia Research Articles

AB0136 Induction of reg ia, a new auto-antigen in sjögren’s syndrome patients, in salivary duct epithelial cells by interleukin-6 and -11

BackgroundThe regenerating gene, Reg, was originally isolated from a rat regenerating islet cDNA library, and its human homologue was named REG Iα. Reg gene product acts as a growth factor...

An important role of Tyk2 in APC function of dendritic cells for priming CD8+ T cells producing IFN‐γ

Tyrosine kinase 2 (Tyk2) contributes to the signals triggered by IL-12 for IFN-gamma production by NK cells and T cells. We found in this study that Tyk2-deficient (-/-) mice showed increased susceptibility at the early stage after an i.p. infection with Listeria monocytogenes, accompanied by impaired IFN-gamma production. The numbers of both MHC class Ib (H2-M3)- or MHC class Ia (Kb)-restricted CD8+ T cells producing IFN-gamma and exhibiting cytotoxicity were significantly decreased in Tyk2-/- mice after infection with L. monocytogenes. Using an adoptive transfer system of OT-I cells expressing OVA(257-264)/Kb-specific TCR into Tyk2-/- mice followed by challenge with recombinant L. monocytogenes expressing OVA, we found that the defective Tyk2 signaling in the host environment was at least partially responsible for the impaired CD8+ T cytotoxic-1 (Tc1) cell responses in Tyk2-/- mice following the infection. Adoptive transfer with MHC class Ib- or MHC class Ia-binding peptide-pulsed BM-derived DC from Tyk2-/- mice induced lower levels of the Ag-specific CD8+ Tc1 cells producing IFN-gamma. These results suggest that Tyk2 signaling is also important for DC function in the induction of MHC class Ia- and class Ib-restricted CD8+ Tc1 cells following L. monocytogenes infection.

Induction of classical and nonclassical MHC-I on mouse brain astrocytes by Japanese encephalitis virus

Infection with Flaviviruses upregulates the cell surface expression of MHC-I, MHC-II, ICAM-1 (CD54), VCAM-1 (CD106) and TAP proteins. Although all these studies have been confirmed using West Nile virus and other Flaviviruses, there are few reports that have examined the effects of Japanese encephalitis virus (JEV) infection directly on nonclassical and classical MHC expression in astrocytes. We show in this report that JEV infection of mouse brain astrocytes results in induction of the nonclassical MHC Class Ib genes, H-2T23, H-2Q4 and H-2T10 in addition to MHC-I, Type I ( α/ β) IFNs, TAP-1, TAP-2, Tapasin, LMP-2, LMP-7 and LMP-10 but not IFNγ, CD80, CD86 and MHC-II genes. The increased cell surface expression of these antigens as well as induction of the genes mentioned above as measured by RT-PCR suggests that JEV infection may lead to the induction of classical MHC Class Ia as well as nonclassical MHC Class Ib molecules.

Laser microdissection and pressure-catapulting technique to study gene expression in the reoxygenated myocardium

For focal events such as myocardial infarction, it is important to dissect infarction-induced biological responses as a function of space with respect to the infarct core. Laser microdissection pressure catapulting (LMPC) represents a recent variant of laser capture microdissection that enables robot-assisted rapid capture of catapulted tissue without direct user contact. This work represents the maiden effort to apply laser capture microdissection to study spatially resolved biological responses in myocardial infarction. Infarcted areas of the surviving ischemic-reperfused murine heart were identified using a standardized hematoxylin QS staining procedure. Standard staining techniques fail to preserve tissue RNA. Exposure of the tissue to an aqueous medium (typically used during standard immunohistochemical staining), with or without RNase inhibitors, resulted in a rapid degradation of genes, with approximately 80% loss in the 1st h. Tissue elements (1 x 10(4)-4 x 10(6) microm(2)) captured from infarcted and noninfarcted sites with micrometer-level surgical precision were collected in a chaotropic RNA lysis solution. Isolated RNA was analyzed for quality by microfluidics technology and reverse transcribed to generate high-quality cDNA. Real-time PCR analysis of the cDNA showed marked (200- and 400-fold, respectively) induction of collagen Ia and IIIa at the infarcted site compared with the noninfarcted site. This work reports a sophisticated yet rapid approach to measurement of relative gene expressions from tissue elements captured from spatially resolved microscopic regions in the heart with micrometer-level precision.

Effects of Age and Gonadal Steroids on the Localization of Antigen Presenting Cells in the Epididymis of the Male Chicken, Gallus domesticus

The goal of this study was to localize antigen presenting cells (APC), which may play roles in defense against pathogens and fertility, and examine the effects of age and gonadal steroids on their population in the rooster epididymis. Healthy White Leghorn male birds (immature 60-day-old birds; matured 150-, 330-, and 550-day-old), and immature birds treated with testosterone propionate (TP) or estradiol benzoate (EB) for 3 or 6 days were used. Cryostat sections of the epididymis and ductus deference were immunostained for Ia to identify APC. RT-PCR was performed to confirm the expression of major histocompatibility complex class II (MHC class II) mRNA in the epididymis. Ia+ cells were localized in the surface epithelium and subepithelial layer of the ductules and occasionally in the luminal content of the epididymis and ductus deference. RT-PCR analysis confirmed expression of MHC class II mRNA in the epididymis, ductus deferens, testis, and spleen. The frequency of Ia+ cells in the subepithelial layer was significantly greater in the proximal efferent ductules than in the other two types of ductules in the epididymis of 550-day-old birds. Although there were no significant differences in the frequencies in the subepithelial layer of the proximal efferent ductules between 60- and 150-day-old birds, the frequencies were significantly greater in 330- and 550-day-old birds than in 60-day-old birds. The frequencies of Ia+ cells in the ductus deferences was increased in the 150-day-old birds compared with the 60-day-old birds, with a larger increase in 330- and 550-day-old birds. The Ia+ cell frequency was significantly increased by EB-injection, but not by TP-injection, on Days 3 and 6 of injection compared with Day 0. These results suggest that the population of APC in the epididymis increases with age after sexual maturation, and estrogen may be one of the factors involved in induction of Ia+ cells.

The expression of MHC class II genes in macrophages is cell cycle dependent.

Using different drugs, we stopped the cell cycle of bone marrow-derived macrophages at different points. After IFN-gamma stimulation, macrophages arrested at the G(1) phase of the cell cycle did not increase cell surface expression of the MHC class II IA. This inhibition is specific, because, under the same conditions, IFN-gamma induces the expression of Fcgamma receptors and the inducible NO synthase mRNA. Treatments that inhibit macrophage proliferation by blocking the cell cycle at the G(1) phase, such as adenosine, forskolin, or LPS, blocked the IFN-gamma induction of IA. Under IFN-gamma treatment, the steady-state levels of IAalpha and IAss mRNA did not increase in cells arrested at the G(1) phase and the half-life of the MHC mRNA was not modified. These data suggest that the cell cycle modulation of IFN-gamma-induced MHC II gene expression occurs at the transcriptional level. The expression of the class II transactivator mRNA induced by IFN-gamma was also blocked when macrophages were arrested at the G(1) phase of the cell cycle, suggesting that the lack of IFN-gamma response occurs at the early steps of MHC class II expression. Finally, macrophages arrested at the G(1) phase showed increased basal levels of cell surface IA due to an increase of the translational efficiency. These data show that the expression of MHC class II genes is regulated by the cell cycle.

Role of muIP-10 in interferon-γ induction of Ia in rat astrocytes

Interferon-γ (IFNγ) is a potent inducer of class II MHC (Ia) in rat astrocytes and microglia which are immunocompetent cells of the central nervous system (CNS). muIP-10, a member of the α-chemokine family, is also induced by IFNγ in these cells. The induction of muIP-10 mRNA occurred in an immediate early manner, while Ia mRNA-induction was delayed and required new protein synthesis. We studied the possible role of muIP-10 in IFNγ-mediated induction of Ia in astrocytes. Antibodies to muIP-10 protein significantly inhibited the expression of surface Ia molecules by astrocytes. Incubation of astrocytes with antisense-oligonucleotides against muIP-10 mRNA also reduced the number of Ia positive cells inducible by IFNγ. Neither the number of IFNγ-inducible class I MHC positive cells nor the number of class I molecules expressed per cell were affected by antisense-oligonucleotides against muIP-10, indicating the specificity of the oligonucleotide and the selective requirement of muIP-10 for Ia induction by IFNγ. Transient transfection of astrocytes with plasmids expressing muIP-10 in the antisense orientation also reduced the number of Ia positive astrocytes. These studies suggest a role for muIP-10 protein as an autocrine factor that enhances the expression of IFNγ-inducible Ia on astrocytes. This could create focal areas rich in Ia expressing cells which could more efficiently present antigens to T cells, leading to immune-mediated inflammation such as in multiple sclerosis.

Components of the protein kinase C pathway induce Ia expression after injection into macrophages.

Macrophages are activated by a variety of microbial and cytokine stimuli. One feature of activation is the induction of class II Ag (Ia) on the cell surface. To understand the intracellular events that occur during activation, we investigated various agents with intracellular activities, and examined their effects on the induction of Ia. We first noted that several agents that activate protein kinase C (PKC) induced Ia, and that several inhibitors of PKC inhibited Ia induction by IFN-gamma. To directly test whether PKC induced Ia, we microinjected normal peritoneal macrophages with this enzyme and other intracellular mediators, then examined Ia expression. We observed that injection of PKC itself, or of other intracellular proteins thought to participate in the PKC pathway (Ras or phospholipase C gamma) strongly induced Ia expression. The Ia-inducing activity of transforming Ras protein was blocked by kinase inhibitor treatment of cells, suggesting that Ras signal transduction requires kinase activity. On the other hand, components of the protein kinase A pathway (phospholipase A2 and protein kinase A itself) did not induce Ia. Thus, the PKC pathway can control expression of macrophage surface Ia, possibly by regulating the genes of the MHC, and may play many other roles in the activation of macrophages.

Suppression of Ia antigen expression on gamma interferon treated macrophages infected with Ehrlichia risticii

Ehrlichia risticii is an obligate intracellular bacterium of monocytes/macrophages. In this report, using immunofluorescence staining, flow cytometry, and Kolmogorov-Smirnov analysis of histograms, the response of P338D 1 and peritoneal macrophages stimulated with recombinant murine interferon-γ (rIFN-γ) was examined for the expression of major histocompatibility complex Class II gene product (Ia) and effect of E. risticii infection on induction of Ia surface expression. Maximal expression of Ia by sham-infected P388D 1 cells was observed 2 days post rIFN-γ addition followed by a progressive decline. These stimulatory effects of rIFN-γ were dose dependent. Relative to sham-infected P388D 1 cells, the induction of Ia by rIFN-γ (200 U ml −1) on E. risticii-infected P388D 1 cells was significantly suppressed at each time point tested through Day 5 with maximal suppression of 88% occurring on Day 2. Similarly, the induction of Ia by rIFN-γ on E. risticii-infected peritoneal macrophages was significantly suppressed by 77% (fluorescent microscopy) when compared to sham-infected peritoneal macrophages. The higher dose of rIFN-γ (2000 U ml −1) failed to restore Ia surface expression by E. risticii-infected P388D 1 cells. The suppression of Ia on P388D 1 cells in response to RIFN-γ was not related to the degree of infection of these cells by E. risticii. A soluble inhibitor substance was not demonstrable in the supernatant from E. risticii-infected cells, nor were inhibitor levels of prostaglandin E 2 levels found in the supernatant. Suppression of surface Ia expression on the macrophage suggests a mechanism whereby I. risticii may evade T-lymphocyte recognition, hinder antigen-specific T-lymphocyte activation, and promote their own survival.

Interleukin 3: From colony-stimulating factor to pluripotent immunoregulatory cytokine

IL-3 is best known as a multicolony-stimulating factor, produced by T-cells, mast cells and eosinophils. Based on its broad spectrum of hemopoietic growth factor activity, the role of IL-3 in the homeostasis of leukocytes is apparent, although the precise mechanisms of this process are yet to be determined. The fact that activated T-helper cells are one of the most potent sources of IL-3 suggests an additional role for IL-3 in the regulation of the immune response. Although the presence of IL-3 receptors on monocytes has been demonstrated, the role of IL-3 in regulating macrophage functions was not clear until recently. We have demonstrated a unique spectrum of macrophage activating properties of IL-3, distinct from that of IFNγ, IL-4 and other CSFs. These novel macrophage activating properties of IL-3 include its capacity to directly induce the expression of Ia antigens and members of the β 2 integrin family (e.g. CD11a/CD18[LFA-1]), as well as to contribute to the regulation of cytokine production (IL-1, IL-6 and TNFα). Moreover, we observed a significant synergy between IL-3 and IFNγ in the induction of Ia and LFA-1, as well as between IL-3 and LPS in the induction of Ia and macrophage cytokines. Our data suggest that IL-3 may help control the antigen presentation (AP) capacity of macrophage via the regulation of Ia, β 2 integrins and macrophage cytokines. It is also possible that the differential state of activation of macrophages that we demonstrated following their induction with IL-3, IFNγ or IL-3 + IFNγ results in the development of functionally distinct AP cells. Further immunoregulatory properties of IL-3 were observed in vivo by Kimoto and colleagues when IL-3 administration led to profound enhancement of the T-cell-dependent immune response, with no changes in the antibody response to T-cell-independent antigens. Thus, in vitro and in vivo data both confer an immunoregulatory role for IL-3 in the development of the immune response, most likely via its effects on the antigen presenting cells (macrophages). This review will summarize evidence documenting a range of immunoregulatory properties of IL-3 and discuss the mechanisms of action of IL-3.

Mechanism of toxicity of the antimelanoma drug 4-hydroxyanisole in mouse hepatocytes

To elucidate the mechanism of the hepatotoxicity of 4-hydroxyanisole (4-HA), its effect on the viability of mouse hepatocytes in suspension was investigated. Cell viability was assessed by measurement of release of lactate dehydrogenase into the medium. 4-HA was cytotoxic in a concentration-dependent and time-dependent fashion with an IC 50 of 0.26 mmol/l after 4 h incubation. Almost all cells were killed after exposure to 4-HA for 4 h at 0.5 mmol/l or for 2 h at 1.0 mmol/l. At 5 and 10 mmol/l, 4-HA caused less cytotoxicity than 1 mmol/l or below. On coincubation with the P450 inhibitor octylamine, 4-HA cytotoxicity was reduced, which suggests the involvement of cytochrome P450 in the hepatocytotoxicity of this drug. Induction of P450 isoenzymes IA, IIB and IIE1 by pretreatment of mice with phenobarbitone, 3-methylcholanthrene or acetone had no significant effect on the toxicity of 4-HA towards hepatocytes. Depletion of hepatic glutathione by pretreatment of mice with buthionine sulphoximine (1.6 g/kg, intraperitoneally) 4 h before cell isolation led to an increase in 4-HA cytotoxicity. Incubation with N-acetylcysteine (10 mmol/l) abolished the cytotoxicity of 4-HA (1 mmol/l). Both these results are consistent with the intermediacy of a reactive metabolite of 4-HA. Production of hydroquinone by oxidative demethylation of 4-HA as toxication mechanism can be excluded as formation of formaldehyde was not observed on incubation of 4-HA with mouse liver microsomes. 3,4-diacetoxyanisole, a prodrug of the known 4-HA metabolite 3,4-dihydroxyanisole, was not more cytotoxic towards hepatocytes than 4-HA. This result suggests that 3,4-dihydroxyanisole is unlikely to mediate the hepatotoxicity of 4-HA.

Ion channels and b cell mitogenesis

Given the presence of ionic channels at the membrane of lymphocytes, we have analyzed the effect of various channels blockers on B lymphocytes activation. TEA and 4-AP, two K + channels blockers, quinine, a blocker of Ca 2+-activated K + channels, nickel and verapamil, two Ca 2+ channels blockers, all inhibited LPS-induced B cell proliferation. However, these drugs neither inhibited the induction of Ia and FcγRII expression nor cell enlargement and early RNA synthesis, indicating that the entry of B lymphocytes into G1 phase was not affected. In contrast, both late RNA synthesis and the induction of the TfR, which occur while the cell progress through G1, were inhibited by these blockers. These data show that TEA, quinine and verapamil block B lymphocyte activation during the G1 phase, probably between G1A and GIB. To question whether these effects were due to the block of voltage-activated K + channels, we compared the ability of TEA, quinine, verapamil, 4-AP and nickel to block proliferation and K + channels. A striking correlation was found for all the drugs but less for 4-AP. Moreover, TMA, a TEA analog unable to block K + currents, did not affect B cell proliferation. Taken together, our data suggests that functional voltage-gated K + channels are required at a precise stage of the G1 phase of the B cell cycle.

Pharmacologic evidence for the requirement of protein kinase C in IFN-induced macrophage Fc gamma receptor and Ia antigen expression.

Previous studies have implicated protein kinase C (PKC) as a mediator in the activation of macrophages by interferons. In order to probe further into the suspected role of protein kinase C in mouse peritoneal macrophage activation, the effects of protein kinase inhibitors in macrophage Fc gamma R and Ia Ag expression were studied. The protein kinase inhibitor, H7, reduced basal levels, and inhibited IFN-alpha-induced expression of Fc gamma R significantly. The concentration of H7 required to inhibit 50% of the Fc gamma R induction was approximately 12 microM, which reflects the previously reported affinity of this compound for PKC in vitro. H7 had only a minimal effect on IFN-gamma-induced Fc gamma R, suggesting different pathways of Fc gamma R induction by the two types of IFN. Ia induction by IFN-gamma was also inhibited by H7, indicating that both types of IFN can utilize PKC to mediate at least part of the signal required for Fc gamma R or Ia expression. HA-1004, a derivative of H7 which possesses high affinity for cyclic nucleotide-dependent protein kinases, but low affinity for PKC, did not alter induction, while H8, a slightly less effective PKC inhibitor than H7, was effective at higher concentrations. Another structurally distinct PKC antagonist, staurosporine, was also effective inhibiting IFN-alpha-induced Fc gamma R and IFN-gamma-induced Ia Ag expression, providing additional evidence that PKC is important. H7 was found to be effective when added as late as several hours after IFN treatment, indicating a prolonged or delayed requirement of PKC for optimal induction of Ia and Fc gamma R by IFN.

1,25-Dihydroxyvitamin D3 Antagonizes Interferon-γ-Induced Expression of Class II Major Histocompatibility Antigens on Thyroid Follicular and Testicular Leydig Cells*

Interferon-gamma (IFN gamma) induces production and expression of major histocompatibility complex class II molecules on both marrow-derived and nonbone marrow-derived cell types. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], a seco-steroid derived from vitamin D3, has previously been reported to enhance such expression alone or together with IFN gamma on a number of monocyte/macrophage tumorigenic lines. In contrast, the present studies have found that 1,25-(OH)2D3 inhibited the ability of IFN gamma to induce class II antigen expression on nontransformed rat thyroid follicular epithelial cells (FRTL-5) and mouse testicular Leydig cells (TM3). Although 1,25-(OH)2D3 inhibited the induction of both IA and IE class II locus products, IFN gamma augmentation of class I major histocompatibility complex antigens was not affected. 1,24-(OH)2D3 and 24,25-(OH)2D3 also inhibited class II induction by IFN gamma. Notably, the relative inhibitory ability of these compounds paralleled the strength of their binding affinities for the 1,25-(OH)2D3 receptor, indicating that this antagonistic effect probably requires receptor-ligand interaction. Other steroid hormones, such as hydrocortisone or testosterone, had no inhibitory effect on IFN gamma-induced class II expression on Leydig cells. Additionally, the failure of indomethacin to reverse the effect of 1,25-(OH)2D3 and the finding that exogenous prostaglandin E2 did not inhibit class II induction in these cells indicated that prostaglandins are probably not responsible for this anti-IFN gamma activity. In total, these results suggest that an endocrinological mediator is capable of inhibiting class II induction on resident endocrine tissue populations and, therefore, could help to diminish local CD4+ T-cell recognition of these cells.

Modulation of Forssman Glycosphingolipid Expression by Murine Macrophages: Coinduction with Class II MHC Antigen by the Lymphokines IL4 and IL6

In contrast to murine spleen MФ, resident peritoneal MФ from healthy mice express very little Forssman glycolipid antigen (Fo). The following experiments suggest that Fo expression by peritoneal M(D may be associated with inflammation. Balb/c and CBA/J mice were given inflammatory stimuli by i.p. injection of live BCG, thioglycollate (TG), Corynebacterium parvum (CP), proteose peptone (PP), or LPS. Control animals received pyrogen-free saline. Expression of Fo and Ia antigen by peritoneal M(D was determined by immunofluorescence after 4 d. Application of TG or CP led to an up to 30-fold increase in Fo +, Ia + double positive MФ over that in control animals. LPS caused mainly an increase in the percentage of doublepositive MФ, whereas no effects were seen in BCG or PP treated animals. To clarify the possible involvement of cytokines in this process and to identify these, the effects of LPS and various cytokines on in vitro induction of Fo and Ia expression were studied in further experiments. LPS, IL6, and IL4 caused induction of up to 15 % Fo + and Ia + MФ after a 4 d culture period. MФ colony stimulating factor (M-CSF) from lung-conditioned medium was also moderately active. IL1, TNF, and IL2 had no influence, whereas IFN-γ only induced Ia. For a successful in vitro induction of Fo and Ia, a prior priming of the mice with PP appeared mandatory. This suggests that only MФ of a certain developmental stage can acquire Fo under the influence of the appropriate cytokines. The data may provide the first evidence for cytokine-mediated modulation of a glycolipid antigen of known chemical structure.

Direct evidence for an intracellular role for IFN-gamma. Microinjection of human IFN-gamma induces Ia expression on murine macrophages.

An intracellular action for IFN-gamma was detected by using microinjection technology. Human IFN-gamma (huIFN-gamma) does not ordinarily act on murine cells because it fails to bind to murine cell surface receptors. However, when huIFN-gamma was microinjected into murine macrophages, a time and dose-dependent induction of Ia was detected by autoradiography on the surface of injected and neighboring cells. These results imply a direct role for internalized IFN-gamma and show that huIFN-gamma, although it fails to be recognized by murine cell surface receptors, can act internally on murine cells. The effect on Ia gene expression induced by microinjected huIFN-gamma was in part indirect: granulocyte/macrophage-CSF (GM-CSF) was released by IFN-gamma-injected macrophages, and this secondary mediator appeared to induce Ia on neighboring cells, inasmuch as anti-GM-CSF blocked Ia induction. Anti-GM-CSF also partially blocked Ia induction by extracellular murine IFN-gamma on murine macrophages. Thus, at least some of the Ia induction attributed to IFN-gamma was mediated by GM-CSF.

Regulation of MHC class II gene expression in macrophages by hematopoietic colony-stimulating factors (CSF). Induction by granulocyte/macrophage CSF and inhibition by CSF-1.

CSF-1 and granulocyte/monocyte CSF (GM-CSF) were shown to modulate the levels of Ia gene and protein expression in bone marrow-derived macrophages (BMM). Recombinant GM-CSF induced high levels of Ia expression, similar to the levels induced by INF-gamma, while IL-3 had no effect. In contrast, recombinant CSF-1 not only suppressed the basal levels of Ia gene and protein expression in BMM, but also inhibited the induction of Ia by IFN-gamma and GM-CSF. Basal levels of Ia were not inhibited by recombinant CSF-1 until after 16-24 h of culture, suggesting an indirect mechanism of suppression. IFN-alpha/beta and PGE2 were shown not to be involved in the CSF-1 inhibition of basal levels of Ia expression. However, the CSF-1-mediated suppression of both the basal levels of Ia expression and the induction of Ia in BMM by IFN-gamma and GM-CSF did correlate with the induction of cellular proliferation. These data imply that in addition to regulating hematopoiesis, CSFs may regulate the initiation of the immune response through their effects on Ia expression in macrophages.

PREGNANCY AS A NATURAL MODEL OF ALLOGRAFT TOLERANCE

From an immunologic viewpoint, the fetus with its paternal antigens may be considered a successful allograft in the maternal host. Understanding the basis of this host-allograft relationship remains a fundamental unsolved problem in transplantation immunobiology. We have previously demonstrated that local immunoregulation in the murine placenta prevented macrophage activities necessary for an effective response against the intracellular bacterium Listeria monocytogenes. Given the central role of macrophages both as antigen-presenting and cytolytic effector cells, such local immuno-regulation may ordinarily help prevent rejection of the fetoplacental unit with its paternal alloantigens by the maternal immune system. We therefore examined two types of interaction between macrophages and the placental cells that populate the maternal-fetal interface. (1) Upon activation to kill listeria efficiently, macrophages also acquire cytolytic capacities against some tumor and embryonic cells. We tested the hypothesis that macrophage activation in the placenta was inhibited to prevent macrophages from lysing fetal trophoblasts. We found, however, that trophoblasts isolated by dispase dispersion, differential isopyknic centrifugation, and adherence were not lysed by three different populations of cytolytic macrophages: (a) those activated in vivo during listeriosis, (b) peptone-elicited macrophages activated in vitro by recombinant interferon gamma and other lymphokines, and (c) the macrophage cell line RAW 264.7 activated in vitro. (2) Previous studies had demonstrated that cells from the placental region and their conditioned media inhibited a variety of lymphocyte functions. However, we found that these did not inhibit activation of adherent macrophages as assessed by induction of cell-surface Ia and acquisition of tumoricidal activity. In addition, under conditions where placental cells inhibited the proliferative response of a cloned CD4+ anti-Listeria T cell line to fixed, antigen-pulsed macrophages, the secretion of macrophage-activating lymphokines was not affected. These studies are important because they indicate that previously described suppressor systems in the murine placental region do not account for the profound local deficits in macrophage function seen during listeriosis.

Inducibility of Ia molecules on keratinocytes reflects genetic control on astrocytes in the brain

We have recently observed strain differences in interferon-γ (IFN-γ) induction of Ia on astrocytes in rats and mice that are susceptible or resistant to experimental allergic encephalomyelitiss (EAE) (Massa et al., 1987). We now found that keratinocytes cultured from rat skin show the same strain differences as astrocytes. Keratinocytes of EAE-susceptible Lewis rats are induced to higher levels of Ia expression compared to relatively resistant Brown-Norway (BN) rats, independent of the dose of IFN-γ applied. This difference in regulation is specific for class II histocompatibility molecules because no such differences are seen with class I antigen induction by IFN-γ. That keratinocytes reflect similar strain differences as astrocytes may be explained by common ectodermal origin of these epithelial-like cells. This observation allows the determination of the genetic predisposition of an autoimmune response in the central nervous system by skin biopsy. This approach may be of importance in studies of immunopathological phenomena of brain tissue in man such as multiple sclerosis.

Lymphocyte subsets differentially induce class II human leukocyte antigens on allogeneic microvascular endothelial cells.

Increased expression of major histocompatibility complex class II (Ia) antigens on vascular endothelium is a common observation in allografts undergoing acute rejection. This phenomenon is generally ascribed to the host immune response directed against graft alloantigens, but its cellular and molecular basis are incompletely understood. In the present study we show that constitutively Ia-negative human microvascular endothelial cells (EC) can be induced to express surface class II human leukocyte antigens shortly after exposure to allogeneic lymphocytes in vitro. CD16+ (natural killer) and CD8+ (cytotoxic/suppressor) lymphocytes were efficient in triggering Ia antigen expression by EC, whereas CD4+ (helper/inducer) lymphocytes induced EC Ia expression only if cultured in the presence of autologous monocytes. Binding of lymphocytes to EC was shown to be essential for the subsequent induction of EC Ia, and anti-CD18 (LFA-1) antibody, which blocks lymphocyte-EC adhesion, was the only antibody of a panel of antilymphocyte antibodies that completely blocked the induction of EC Ia. Antibodies to interferon-gamma, which is a potent inducer of EC Ia, and to the CD3 T cell-surface antigen partly inhibited the induction of EC Ia by T cells, but neither antibody had any effect on Ia induction mediated by CD16+ cells, suggesting that T cells and natural killer cells utilize different mechanisms to induce Ia on EC. When combined with data from other laboratories indicating that Ia+ but not Ia- EC stimulate allogeneic T cell proliferation and cytotoxicity, our results suggest that the binding of EC by lymphocyte subpopulations followed by the induction of Ia antigen may represent the initial stage of incompatible allograft rejection.