Surface Ia Research Articles

SUMOylating Two Distinct Sites on the A-type Potassium Channel, Kv4.2, Increases Surface Expression and Decreases Current Amplitude.

Post-translational conjugation of Small Ubiquitin-like Modifier (SUMO) peptides to lysine (K) residues on target proteins alters their interactions. SUMOylation of a target protein can either promote its interaction with other proteins that possess SUMO binding domains, or it can prevent target protein interactions that normally occur in the absence of SUMOylation. One subclass of voltage-gated potassium channels that mediates an A-type current, IA, exists as a ternary complex comprising Kv4 pore-forming subunits, Kv channel interacting proteins (KChIP) and transmembrane dipeptidyl peptidase like proteins (DPPL). SUMOylation could potentially regulate intra- and/or intermolecular interactions within the complex. This study began to test this hypothesis and showed that Kv4.2 channels were SUMOylated in the rat brain and in human embryonic kidney (HEK) cells expressing a GFP-tagged mouse Kv4.2 channel (Kv4.2g). Prediction software identified two putative SUMOylation sites in the Kv4.2 C-terminus at K437 and K579. These sites were conserved across mouse, rat, and human Kv4.2 channels and across mouse Kv4 isoforms. Increasing Kv4.2g SUMOylation at each site by ~30% produced a significant ~22%–50% decrease in IA Gmax, and a ~70%–95% increase in channel surface expression. Site-directed mutagenesis of Kv4.2g showed that K437 SUMOylation regulated channel surface expression, while K579 SUMOylation controlled IA Gmax. The K579R mutation mimicked and occluded the SUMOylation-mediated decrease in IA Gmax, suggesting that SUMOylation at K579 blocked an intra- or inter-protein interaction involving K579. The K437R mutation did not obviously alter channel surface expression or biophysical properties, but it did block the SUMOylation-mediated increase in channel surface expression. Interestingly, enhancing K437 SUMOylation in the K579R mutant roughly doubled channel surface expression, but produced no change in IA Gmax, suggesting that the newly inserted channels were electrically silent. This is the first report that Kv4.2 channels are SUMOylated and that SUMOylation can independently regulate Kv4.2 surface expression and IA Gmax in opposing directions. The next step will be to determine if/how SUMOylation affects Kv4 interactions within the ternary complex.

Serum-derived MHC class II molecules: Potent regulators of the cellular and humoral immune response

Soluble MHC antigens are detected in body fluids but their role and origin are still unclear. This study examined whether serum IA antigens, isolated from BALB/c mice (sIA d), could modulate the immune response. Specific purification procedures isolated intact IA molecules, which were thereafter applied to functional assays. Thus, sIA d were shown to stimulate spleen cell proliferation and the major target was identified to be the CD4 + cell population. Inhibition of the CD4 co-receptor using specific neutralizing antibodies destroyed the sIA d-mediated proliferative activity, while sIA d successfully antagonized surface IA d antigens for binding to anti-IA d antibody. Serum-IA d stimulated BALB/c versus C3H/HeN but not C3H/HeN versus BALB/c mixed lymphocyte reactions, while increasing responsiveness to Legionella pneumophila. However, sIA d displayed an inhibitory activity during the effector phase of the humoral response, since they inhibited the anti-DNP-specific IgM production to a DNP–HSA hapten–carrier system. Furthermore, sIA d molecules increased Th1/Th2 cytokines during the L. pneumophila stimulus, while decreasing IL-2, GM-CSF and increasing IL-4, IL-15 during the DNP–HSA stimulus. These results suggest that sIA d, following steps similar to surface class II antigen binding mechanisms, stimulate the initiation of a humoral or cellular immune response but rather inhibit the effector phase of the reactions, attributing thus soluble class II MHC antigens important immunomodulatory roles.

Characterization of a Novel H2A−E+ Transgenic Model Susceptible to Heterologous but Not Self Thyroglobulin in Autoimmune Thyroiditis: Thyroiditis Transfer with Vβ8+ T Cells

Recently we reported on a novel H2E transgenic, IA-negative model of experimental autoimmune thyroiditis (EAT) that excludes reactivity to self in its susceptibility pattern to heterologous thyroglobulin (Tg). In conventional, susceptible mouse strains, EAT is inducible with both homologous and heterologous Tg; e.g., human (h)Tg shares conserved thyroiditogenic epitopes with mouse (m)Tg. However, when an H2Eak transgene is introduced into class II-negative B10.Ab0 mice, which express neither surface IA (mutant Aβ-chain) nor surface IE (nonfunctional Ea gene), the resultant H2Eb molecules are permissive for EAT induction by hTg, but not self mTg. Also, the hTg-primed cells do not cross-react with mTg.To explore this unique capacity of E+B10.Ab0 mice to distinguish self from nonself Tg, we have developed T cell lines to examine the T cell receptor repertoire and observed a consistent Vβ8+ component after repeated hTg stimulation. Enrichment and activation of Vβ8+ T cells by either superantigen staphylococcal entertoxin B or anti-Vβ8 in vitro enabled thyroiditis transfer to untreated A−E+ recipients, similar to hTg activation. Vβ8+ T cells isolated by FACS from hTg-immunized mice also proliferated to hTg in vitro. These studies support the contribution of Vβ8 genes to the pathogenicity of hTg in this H2A−E+ transgenic model.

Electrochemical, spectroscopic and SPM evidence for the controlled formation of self-assembled monolayers and organised multilayers of ferrocenyl alkyl thiols on Au(111)

Organised multilayers were formed from the controlled self-assembly of ferrocene alkyl thiols on Au(111) surfaces. The control was accomplished by increasing the concentration of the thiol solutions used for the assembly. Cyclic voltammetry, ellipsometry, scanning probe microscopy (STM and AFM) and in situ FTIR spectroscopy were used to probe the differences between mono- and multilayers of the same compounds. Electrochemical desorption studies confirmed that the multilayer structure is attached to the surface ia one monolayer. The electrochemical behaviour of the multilayers indicated the presence of more than one controlling factor during the oxidation step, whereas the reduction was kinetically controlled which contrasts with the behaviour of monolayers, which exhibit kinetic control for the oxidation and reduction steps. Conventional and imaging ellipsometry confirmed that multilayers with well-defined increments in thickness could be produced. However, STM indicated that at the monolayer stage, the thiols used promote the mobility of Au atoms on the surface. It is very likely that the multilayer structure is held together through hydrogen bonding. To the best of out knowledge, this is the first example of a controlled one-step growth of multilayers of ferrocenyl alkyl thiols using self-assembly techniques.

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.

Regulation of langerhans cell function via blood borne factor(s)

Langerhans cells (LC) are epidermal dendritic cells that are functionally labile. Freshly obtained LC (fLC) readily activate allogeneic T cells, but they are incapable of activating autologous T cells; and even when pulsed with antigen, they fail to activate naive, antigen-specific T cells. When fLC are cultured for 2–3 days in the presence of keratinocytes, LC swiftly up-regulate surface expression of class I and II MHC molecules, and express de novo the co-stimulatory molecules B7 and ICAM-1. In addition to displaying enhanced ability to activate allogeneic T cells, cultured LC acquire the novel functional property of activating autologous T cells. It is believed that keratinocytederived GM-CSF is the primary driving force responsible for the conversion of fresh to cultured LC in vitro. However, in vivo administration of GM-CSF, either intracutaneously or systematically, fails to induce LC to undergo functional transformation in situ. Moreover, despite a high level of GM-CSF in the circulation, fLC from mice bearing GM-CSF-producing tumors display no ability to activate syngeneic T cells. These observations suggest that a homeostatic factor that antagonizes the effect of GM-CSF may be present in vivo. To test this possibility, we have examined the functional properties of LC prepared from mouse skin that had been explanted in vitro for 3 days. We found that the functional and phenotypic features of these cells closely resembled those of LC cultured in single cell suspension: the cells strongly expressed B7-1 and B7-2, and displayed enhanced expression of class II MHC molecules; they readily activated naive autologous T cells. Strikingly, when explants or suspensions of fresh epidermal cells were cultured in the presence of 10% mouse serum they failed to acquire syngeneic T cell activating properties; and surface up-regulation of Ia and de novo expression of B7 was inhibited. However, the cultured cells still expressed surface Ia and readily activated allogeneic naive T cells. If mouse serum was added only during the last 24 h of culture, the LC displayed full functional transformation. Human, rabbit and bovine serum showed no inhibitory effect on mouse LC. Our data suggest that mouse serum contains a factor (or factors) that inhibits, in a species-specific manner, epidermal LC from undergoing functional transformation in vitro, and this factor may maintain epidermal LC in the ‘fresh’ functional program in vivo.

Superantigen-Induced Langerhans Cell Depletion Is Mediated by Epidermal Cell-Derived IL-1α and TNFα

The purpose of this study was to examine the role of cytokines in staphylococcal enterotoxin-A (SEA)-induced epidermal Langerhans cell (LC) depletion. This was accomplished by analyzing the effect of SEA on cytokine secretion by cultured epidermal cell populations by means of ELISA and by assessing the capacity of cytokines and cytokine-specific antibodies to affect epidermal LC density (as measured by immunoperoxidase staining of epidermal cells bearing surface Ia). The results of this study indicate that SEA induces the secretion of IL-1α and TNFα by epidermal cells, that these cytokines induce LC depletion from epidermis, and that antibodies specific for these agents inhibit the depletion of LC by SEA. Taken together, these findings suggest that IL-1α and TNFα may play essential roles in SEA-mediated depletion of LC from murine epidermis.

Peritoneal and Splenic Macrophage Functions in the Tumor-Bearing Host

In the tumor-bearing host, depression of cell-mediated immunity has been well-demonstrated, but little is known about alterations in macrophage functions. We hypothesized that the presence of a tumor may cause functional suppression of peritoneal macrophage and splenic macrophage functions, perhaps due to prostaglandin-E 2 (PGE 2) and nitric oxide. C57 BL/6 mice ( n = 18) were injected subcutaneously with Lewis lung carcinoma 3 tumor. Control mice ( n = 18) received no tumor and were pair-fed to intake of the tumor group. On Day 21, peritoneal and splenic macrophages were harvested. Cell surface Ia expression, cytotoxicity against P815 target cells, Candida albicans killing, and production of PGE 2, nitric oxide, and superoxide were measured. Mean Ia expression was decreased in tumor animals for both peritoneal and splenic macrophages. In tumor animals PGE 2 production significantly increased in both peritoneal and splenic macrophages. Superoxide production significantly decreased in peritoneal macrophages but significantly increased in splenic macrophages. Nitric oxide production was also significantly depressed in tumor-bearing host peritoneal macrophages. C. albicans killing was significantly depressed in tumor animals' peritoneal macrophages but showed little change in splenic macrophages. In tumor animals cytotoxicity was significantly decreased in peritoneal macrophages but significantly increased in splenic macrophages. In the tumor-bearing host, peritoneal macrophages have significantly decreased accessory and effector functions. Splenic macrophages demonstrate decreased accessory but enhanced effector functions. PGE 2, superoxide, and nitric oxide appear to be important mechanisms of altered macrophage function in the tumor-bearing host; modifying their cellular production may enhance host defense.

Entamoeba histolytica suppresses gamma interferon-induced macrophage class II major histocompatibility complex Ia molecule and I-A beta mRNA expression by a prostaglandin E2-dependent mechanism.

The surface expression of class II major histocompatibility complex immune-associated (Ia) antigen is a principal accessory function of macrophages for antigen-specific T-cell activation and immunoregulation. To explore the mechanisms of impaired cell-mediated immunity in invasive amebiasis, we investigated the effects of Entamoeba histolytica proteins on gamma interferon (IFN-gamma)-induced Ia expression by murine bone marrow-derived macrophages. Pretreatment of macrophages with secreted (conditioned medium) or whole soluble amebic proteins inhibited the induction of IFN-gamma-induced surface Ia antigen expression by 30 to 61% but had no effect on surface Ia molecules already expressed. By Northern (RNA) blot analysis, amebic proteins suppressed IFN-gamma-induced macrophage I-A beta mRNA accumulation by 36% but did not alter the constitutive levels of actin mRNA expression. E. histolytica stimulated macrophages to produce high levels of prostaglandin E2 (PGE2) as determined by reverse-phase high-pressure liquid chromatography and quantification by PGE2-specific radioimmunoassay. Inhibition of PGE2 biosynthesis with the cyclooxygenase inhibitor indomethacin abrogated ameba-induced suppression of Ia antigen by 60%, whereas exogenously added PGE2 decreased IFN-gamma-induced macrophage Ia expression by 44%. Our results suggest that the mechanism whereby E. histolytica suppresses IFN-gamma-induced macrophage surface Ia molecule synthesis and I-A beta mRNA expression is by stimulating the production of PGE2, which acts in an autocrine fashion for immunoregulation. E. histolytica subverting critical macrophage accessory function via PGE2 biosynthesis is a novel strategy which the parasite uses to suppress host defenses.

Modulation of airway intraepithelial dendritic cells following exposure to steroids.

Recent studies from our laboratory have identified a network of constitutively class II major histocompatibility complex (MHC) (Ia)-bearing dendritic cells (DC) within the epithelium of the conducting airways of laboratory animal species and in humans. These studies have also demonstrated that the density of the DC network increases within the airway epithelium in response to inflammatory challenge. In the present report, we demonstrate that exposure of adult rats to inhaled steroids leads to a rapid but readily reversible decrease both in the number of airway intraepithelial DC, and in their surface Ia expression. Similar changes are also seen in response to high doses of systemic dexamethasone. In addition, we demonstrate that steroid inhalation reduces the rate of postnatal expansion of the airway intraepithelial DC network in rat pups, and prevents the rapid expansion of the DC network in adults which occurs during the acute inflammatory response following inhalation of microbial stimuli.

Phenotypic and Ultrastructural Properties of Antigen-Presenting Cells Involved in Contact Sensitization of Normal and UV-Irradiated Mice

We investigated the surface phenotype and localization of hapten in antigen-presenting cells involved in the induction of contact hypersensitivity or tolerance. Dendritic cells collected 18 h earlier from the draining lymph nodes of mice sensitized with fluorescein isothiocyanate (FITC), which induce contact hypersensitivity upon injection into the foot-pads of naive mice, stimulated proliferation of lymphocytes from FITC-specific T-cell lines. By immunoelectron microscopy, these cells expressed high amounts of surface Ia molecules but had a negligible amount of FITC on the cell membrane. The majority of the FITC was localized in discrete structures in the cytoplasm, including mitochondria, endocytic vesicles, lysosomes, and cored tubules. In contrast, lymph node cells conjugated with FITC in vitro did not stimulate proliferation of FITC-specific T cells and showed a heavy, uniform distribution of FITC throughout the cytoplasm. Draining lymph node cells from mice exposed to ultraviolet (UV) radiation and then sensitized by applying FITC to the UV-irradiated skin, which induce tolerance upon injection into naive mice, induced significantly less proliferation of FITC-specific T cells than draining lymph node cells from unirradiated mice. The differences in activity of these cells, relative to draining lymph node cells from unirradiated, FITC-sensitized mice could not be attributed to a decreased number of Ia+ dendritic cells in the DLN, decreased surface expression of Ia molecules on these cells, or an alteration in the intracellular localization of hapten. However, a significantly higher percentage of the FITC+ dendritic cells from UV-irradiated mice expressed mac-1, -2, and -3 and F4/80 macrophage markers than did those from unirradiated animals, and fewer cells contained Birbeck granules, suggesting that a different population of Ia+ antigen-presenting cells may reach the draining lymph nodes of UV-irradiated mice.

Down regulation of Ia expression in macrophages following incubation with mycobacteria.

Macrophages are known to release cytokines in response to various kinds of stimulators. In the present study, peritoneal macrophages from C3H/He or C3H/HeJ mice were incubated in vitro with heat-killed M. lepraemurium, M. intracellulare or M. gordonare for 3 days followed by harvest culture supernatant to analyze cytokine activities. It, therefore, seems that macrophages phagocytizing these mycobacteria, released interleukin-1 (IL-1) and tumor necrosis factor (TNF) in culture media. The amount of release was dose dependent on mycobacteria employed. In addition, macrophages, as already have reported elsewhere, treated with IFN for 2 to 3 days showed enhanced expression of surface Ia; although the expression was inhibited if the cells phagocytized mycobacteria. Similarly, the reduced expression of Ia was observed in peritoneal macrophages from MRL/lpr mice after 3 day-culture with mycobacteria in vitro. More importantly, in the presence of the supernatant obtained from macrophages incubated with mycobacteria, IFN gamma-treated normal macrophages exhibited suppressed expression of Ia. These results demonstrate that cytokine release and reduced expression of surface Ia in macrophages are simultaneous phenomena after phagocytosis of mycobacteria. Suppression of Ia may be in part induced by Ia suppressive factor(s) released from mycobacterium-phagocytized macrophages.

Splenic macrophage activation and functions in amyloid enhancing factor-induced secondary amyloidosis Study of phagocytosis, killing, respiratory burst, and MHC class II surface expression

Secondary amyloidosis is a systemic disease characterized by the extracellular tissue deposition of insoluble fibrillar amyloid A protein. Aberrant metabolism of serum amyloid A protein by reticuloendothelial cells is thought to result in the accumulation of fibrils within the tissue. Treatment of mice with amyloid-enhancing factor (AEF) in conjunction with an inflammatory stimulus (i.e., AgNO3) induced amyloid deposition within 48-72 h. The activation state of a macrophage largely defines its enzymatic capabilities. In the studies reported here, we examined the effect of AEF on spleen macrophage activation using both functional and phenotypic assays. We found that while AEF in the presence or absence of AgNO3 has no apparent effect on the ability of spleen and liver macrophages to phagocytose or kill Listeria monocytogenes, it appears to block enhanced respiratory burst function (as measured by O2- production) observed with AgNO3 alone. AEF therefore seems capable of inhibiting certain macrophage activation-associated functions while not affecting others. Our activation phenotype studies, using surface Ia expression, reveal that AEF blocks the increase in number of splenic macrophages expressing Ia seen with AgNO3 alone. Treatment with interferon-gamma was found to restore decreased Ia expression in animals given AEF+AgNO3 but did not prevent amyloid A fibril deposition.

Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages.

Class II major histocompatibility complex (Ia)-bearing dendritic cells (DC) from airway epithelium and lung parenchyma express low-moderate antigen presenting cell (APC) activity when freshly isolated. However, this function is markedly upregulated during overnight culture in a manner analogous to epidermal Langerhans cells. The in vitro "maturation" process is inhibited by coculture with pulmonary alveolar macrophages (PAM) across a semipermeable membrane, and the degree of inhibition achieved can be markedly increased by the presence of tumor necrosis factor alpha. In addition, PAM-mediated suppression of DC function is abrogated via inhibition of the nitric oxide synthetase pathway. Functional maturation of the DC is accompanied by increased expression of surface Ia, which is also inhibited in the presence of PAM. Prior elimination of PAM from DC donors via intratracheal administration of the cytotoxic drug dichloromethylene diphosphonate in liposomes, 24-72 h before lung DC preparation, achieves a comparable upregulation of APC activity, suggesting that (consistent with the in vitro data) the resident PAM population actively suppresses the APC function of lung DC in situ. In support of the feasibility of such a regulatory mechanism, electron microscopic examination of normal lung fixed by intravascular perfusion in the inflated state (which optimally preserves PAM in situ), revealed that the majority are preferentially localized in recesses at the alveolar septal junctions. In this position, the PAM are in intimate association with the alveolar epithelial surface, and are effectively separated by as little as 0.2 microns from underlying interstitial spaces which contain the peripheral lung DC population. A similar juxtaposition of airway intraepithelial DC is demonstrated with underlying submucosal tissue macrophages, where the separation between the two cell populations is effectively the width of the basal lamina.

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.

The effects of MHC chromosome dosage on bursal B-cell subpopulations in embryonic and neonatal chickens

The effect of MHC dosage on the population structure of developing bursal lymphocytes was studied in the Trisomic strain of chickens. These chickens contain either 2 (disomic), 3 (trisomic), or 4 (tetrasomic) copies of the MHC-encoding chromosome. To assess the developmental profile of bursal cell populations, bursal cells were characterized in the older embryo (18 days of incubation) and at two neonatal ages (1 day and 1 week post hatch) by a number of cellular parameters. Both transient and persistent alterations in cellular composition were revealed in the bursal subpopulations of trisomics and tetrasomics (aneuploids). Aneuploid bursal cells showed increased cell surface expression of MHC class II Ia antigen, at each age, compared to normal disomic cells. Aneuploid cell populations showed an increased incidence of small cells, at each age, a decreased incidence of Ia+ and IgM+ cells during late embryogenesis, and an increased incidence of IgG+ cells in the late embryo and 1-day-old chick. Significantly fewer proliferating cells were observed in the aneuploids, as compared to disomics, in the late embryo and 1-week-old chick. Three subpopulations were observed to be persistently altered in the MHC-aneuploids: an increased pool of Ia+++, small, IgM+ cells; a decreased pool of Ia+, large, IgM+ cells; and a decreased pool of Ia+, large, IgM- cells. Thus, increasing MHC-chromosome dosage is accompanied by specific alterations in B-cell differentiation. The elevated surface Ia antigen expression found on developing aneuploid B cells may play a role in these alterations.

Spleen dendritic cells exhibit altered morphology and increased allostimulatory capacity after short-term culture.

Ia-bearing dendritic cells (DC) are a class of bone marrow-derived antigen-presenting cells that appear to possess an increased capacity to stimulate resting T lymphocytes. DC from different tissues share several morphologic, phenotypic and functional attributes. For example, freshly isolated DC from spleen resemble phenotypically and functionally freshly isolated Langerhans cells (LC) from epidermis; in addition, during short-term culture both DC and LC undergo several parallel changes including modifications affecting phenotype, capacity to present protein antigens, and ability to route surface Ia molecules into intracellular acidic compartments (J Immunol 1990: 145: 2820-2826). In the present study we show, using immunoelectron microscopy with anti-Ia and anti-33D1 monoclonal antibodies, freshly isolated DC in suspension to have a smooth cell surface with few and short cytoplasmic projections. By contrast, cultured DC display conspicuous bulbous cytoplasmic protrusions. In addition, spleen DC following culture for 24-48 hours exhibit an increased ability to stimulated allogeneic T lymphocytes in the primary mixed leukocyte reaction. These changes, similar to those described for freshly isolated and cultured LC respectively, further substantiate the close relationship between DC and LC.

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.

Functional role of self IA molecules in polyclonal B cell activation using an autoreactive B cell clone derived from (NZB X NZW) F 1 mice

The mechanism of polyclonal B cell activation in autoimmune diseases was investigated by using an autoreactive B cell clone established by somatic hybridization with B cells derived from NZB X NZW (B/W) F 1 mice. Briefly, splenic B cells from B/W F 1 mice were fused with M12.4.1, a mutant of a B cell line, in the presence of polyethylene glycol and DMSO. NW47.7, a subclone of a resulting hybridoma, expresses B cell surface antigens on the cell membrane, namely IA d, IgM, B220, the receptors for the C3 fragment of complement (C3R), and the Fc portion of IgG (FcγR). It also possesses a receptor molecule for mouse red blood cells treated with bromelain (Br-MRBC) on its surface, by rosette-forming assay with Br-MRBC. In contrast, parental M12.4.1 does not express IA d and IgM on the cell membrane, and does not bind to Br-MRBC under the same conditions. Thus, it is likely that NW47.7 may be an autoreactive B cell clone specific for Br-MRBC. Interestingly, NW47.7 was induced to generate a significant number of IgM-secreting cells when treated with Br-MRBC and rIL-5. Furthermore, mAb against IA d molecules, but not IA k and K dD d, markedly inhibited the differentiative effect of polyclonal activators such as LPS and rIL-5. Also, when MHC identical irradiated B cells were added to the culture of NW47.7 as a stimulator, the induction of IgM-producing cells was greatly augmented, but this augmenting effect was lost by interfering with direct contact of NW47.7 cells with stimulator B cells using a semipermeable membrane, as well as by the addition of mAb against IA d molecules. In addition, irradiated NW47.7, but not M12.4.1, by itself could enhance the secretion of IgM by NW47.7 as a stimulator, but this enhancing effect markedly decreased in the presence of anti-IA d mAb. The present results suggest that surface IA molecules on B cells are involved during the differentiative response to polyclonal activators, and may directly provide a differentiative signal for maturation of B cells into IgM-secreting cells.