Functional connectivity in depressive, obsessive-compulsive, and schizophrenic disorders: an explorative correlational analysis of regional cerebral metabolism

Cellular elements of the vascular wall, such as endothelium (En) and smooth muscle cells/pericytes (SM/P) possess important immunologic properties. We have previously reported that murine brain microvessel En cells and SM/P express Major Histocompatibility (MHC) class II molecules and activate syngeneic CD4+ T cells in a class II dependent way. Herein we compare MHC class II expression on brain microvessel En to aorta large vessel En cells in order to explore the mechanisms of immune responses in brain tissue versus other peripheral tissues. Interestingly, we demonstrate that En cells from brain microvessel and large aortic vessel express the I-A but not the I-E subunit of MHC class II molecules. The expression of I-A class II molecules can be upregulated on brain microvessel and aortic En cells by interferon-gamma (IFN-gamma). Similarly, the expression of I-A, but not I-E, MHC class II molecules on brain microvessel endothelial cells was upregulated in the presence of activated T cells. Interleukin-10 (IL-10) was found to inhibit IFN-gamma-mediated upregulation of I-A class II molecule expression on aortic but not on microvessel En cells. Our data may indicate that some differences in organ-specific immune responses, are defined by local parameters, such as MHC distribution and regulation.     

IFN-alpha priming of human monocytes differentially regulates gram-positive and gram-negative bacteria-induced IL-10 release and selectively enhances IL-12p70, CD80, and MHC class I expression

Administration of IFN-gamma and IFN-alpha may protect or induce autoimmune diseases. Although the in vitro regulation of monokine secretion by IFN-gamma have been extensively studied, the regulatory function of IFN-alpha has not yet been elucidated. We compared IFN-alpha and IFN-gamma, added alone or simultaneously before bacterial stimulation, for the control of monokine release and the expression of costimulatory molecules by human monocytes. Our data show that: 1) IFN-alpha primes monocytes for increased production of IL-10 in response to Staphylococcus aureus Cowan I strain (SAC) but not to LPS, leading to a lack of IFN-alpha priming for TNF-alpha secretion; 2) pretreatment of monocytes with IFN-alpha inhibits LPS- or SAC-induced IL-12p40 production but unexpectedly enhances the release of the biologically active form of IL-12 (IL-12p70); 3) IFN-alpha and IFN-gamma exert an antagonistic effect on LPS- and SAC-induced IL-10 as well as IL-12p40 release, whereas they further enhance IL-12p70 production when added simultaneously; 4) in contrast to IFN-alpha, IFN-gamma primes monocytes to enhance LPS- or SAC-induced TNF-alpha and IL-12 production, but surprisingly, it increases IL-10 production by monocytes following LPS but not SAC stimulation; and finally, 5) IFN-alpha pretreatment selectively up-regulates CD80 and MHC class I expression on monocytes. It is proposed that the outcome of the immune response at the site of inflammation may depend on both the type of bacterial injury (gram-positive or -negative) and of locally produced IFNs, and that the differential and opposite effects of type I and type II IFNs on monocytes may account for the beneficial or detrimental effects of IFN-alpha therapy.     

Deficient major histocompatibility complex class II antigen presentation in a subset of Hodgkin's disease tumor cells

Hodgkin's disease is a common malignancy of the lymphoid system. Although the scarce Hodgkin and Reed-Sternberg (HRS) tumor cells in involved tissue synthesize major histocompatibility complex (MHC) class II and costimulatory molecules such as CD40 or CD86, it is unclear whether these tumor cells are operational antigen-presenting cells (APC). We developed an immunofluorescence-based assay to determine the number of MHC class II molecules present on the surface of single living HRS cells. We found that in fresh Hodgkin's disease lymph node biopsies, a subset of HRS cells express a substantial number of surface MHC class II molecules that are occupied by MHC class II-associated invariant chain peptides (CLIP), indicating deficient loading of MHC class II molecules with antigenic peptides. Cultured Hodgkin's disease-derived (HD) cell lines, however, were found to express few MHC class II molecules carrying CLIP peptides on the cell surface and were shown to generate sodium dodecyl sulphate (SDS)-stable MHC class II alphabeta dimers. In addition to showing deficient MHC class II antigen presentation in a subset of HRS cells, our results show that the widely used HD-cell lines are not ideal in vitro models for the disease. The disruption of MHC class II-restricted antigen presentation in HRS cells could represent a key mechanism by which these tumor cells escape immune surveillance.     

Invariant chain cleavage and peptide loading in major histocompatibility complex class II vesicles

B lymphocytes contain a novel population of endocytic vesicles involved in the transport of newly synthesized major histocompatibility complex (MHC) class II alpha beta chains and alpha beta peptide complexes to the cell surface. We now present evidence that these class II-enriched vesicles (CIIV) are also likely to be a site for the loading of immunogenic peptides onto MHC molecules. We used the serine protease inhibitor leupeptin to accumulate naturally occurring intermediates in the degradation of alpha beta-invariant chain complexes and to slow the intracellular transport of class II molecules. As expected, leupeptin caused an accumulation of Ii chain and class II molecules (I-A(d)) in endosomes and lysosomes. More importantly, however, it enhanced the selective accumulation of a 10-kD invariant chain fragment associated with sodium dodecyl sulfate (SDS)-labile (empty) alpha beta dimers in CIIV. This was followed by the dissociation of the 10-kD fragment, formation of SDS-stable (peptide-loaded) alpha beta dimers, and their subsequent appearance at the cell surface. Thus, CIIV are likely to serve as a specialized site, distinct from endosomes and lysosomes, that hosts the final steps in the dissociation of invariant chain from class II molecules and the loading of antigen-derived peptides onto newly synthesized alpha beta dimers.     

Effects of dietary supplementation with alpha-linolenic acid on the phospholipid fatty acid composition and quality of spermatozoa in cockerel from 24 to 72 weeks of age

The physiopathology of experimental cerebral malaria (CM), an acute neurological complication of Plasmodium berghei ANKA (PbA) infection, involves interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), two cytokines that are known to modulate major histocompatibility complex (MHC) molecule expression. The aim of this study was to evaluate whether the genetic susceptibility to CM is related to the constitutive or IFN-gamma-induced expression of MHC molecules on brain microvessels. To this end, brain microvascular endothelial cells (B-MVEC) were isolated from CM-susceptible (CM-S, CBA/J) and resistant (CM-R, BALB/c) mice. By flow cytometry, we found that less than 5% of CM-S B-MVEC constitutively expressed MHC class I molecules, in contrast to up to 90% of CM-R B-MVEC. Upon stimulation with IFN-gamma, the percentage of positive cells for MHC class I molecules in CM-S B-MVEC became comparable to CM-R B-MVEC, but a higher fluorescence intensity existed on CM-S B-MVEC compared with CM-R B-MVEC. MHC class II molecules were not constitutively expressed on B-MVEC from either strain. IFN-gamma-induced expression of MHC class II (I-A, I-E) molecules was significantly higher in CM-S than CM-R B-MVEC both in percentage of positive cells and fluorescence intensity. These data demonstrate that absent or low MHC class I and higher inducibility of MHC class II expression on B-MVEC are associated with the genetic susceptibility to CM.     

Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.     

DR/CLIP (class II-associated invariant chain peptides) and DR/peptide complexes colocalize in prelysosomes in human B lymphoblastoid cells

In APCs, MHC class II molecules (MHC class II) bind antigenic peptides after HLA-DM mediated removal of CLIP. To characterize intracellular sites of peptide loading in human B lymphoblastoid cell lines, we conducted immunoelectron microscopy studies with Abs recognizing MHC class II associated with CLIP or bound peptide, respectively, together with Abs to HLA-DM and endocytic markers. The distribution of these molecules indicates that peptide binding occurs in compartments with characteristics of normal late endosomes, and in compartments that show characteristics of late endosomes, but are not detectably accessed by endocytosed BSA-gold. The latter compartments may represent or give rise to recycling vesicles that deliver peptide-loaded class II molecules to the cell surface. In addition, we have compared cells in which HLA-DM and HLA-DR interaction is defective with cells in which this interaction is intact, and find that DM/DR interaction is not required for the proper localization of either molecule to peptide-loading compartments.     

Enhanced interaction of HLA-DM with HLA-DR in enlarged vacuoles of hereditary and infectious lysosomal diseases

Following biosynthesis, class II MHC molecules are transported through a lysosome-like compartment, where they acquire antigenic peptides for presentation to T cells at the cell surface. This compartment is characterized by the presence of HLA-DM, which catalyzes the peptide loading process. Here we report that the morphology and function of the class II loading compartment is affected in diseases with a phenotypic change in lysosome morphology. Swollen lysosomes are observed in cells from patients with the hereditary immunodeficiency Chediak-Higashi syndrome and in cells infected with Coxiella burnetii, the rickettsial organism that causes Q fever. In both disease states, we observed that HLA-DR and HLA-DM accumulate in enlarged intracellular compartments, which label with the lysosomal marker LAMP-1. The distribution of class I MHC molecules was not affected, localizing disease effects to the endocytic pathway. Thus, cellular mechanisms controlling lysosome biogenesis also affect formation of the class II loading compartment. Analysis of cell surface class II molecules revealed that their steady-state levels were not reduced on diseased cells. However, in both disease states, enhanced interaction between HLA-DR and HLA-DM was detected. In the Chediak-Higashi syndrome cells, this correlated with more efficient removal of the CLIP peptide. These findings suggest a mechanism for perturbation of Ag presentation by class II molecules and consequent immune deficiencies in both diseases.     

Expression of galectin-1 in the mouse olfactory system

Mast cells hold a key position in the defensive mechanisms against exogenous intruders. In this study, we investigated whether human mast cells express functional major histocompatibility complex (MHC) class II molecules that can transduce endogenous signals and present staphylococcal enterotoxin A (SEA) to T cells. Similar to HMC-1 human mast cell line, umbilical cord blood-derived mast cells express HLA-DR, -DP and -DQ molecules on their surface. MHC class II molecules expressed on HMC-1 cells bind significantly the SEA (a natural MHC class II ligand), and their ligation with specific mAbs or with SEA, leads ultrastructural changes, suggesting their degranulation. Recognition of SEA-bound MHC class II molecules on HMC-1 mast cells by the T cell receptor of K25 cells, an SEA-specific murine T cell hybridoma, triggers significant IL-2 secretion by these T cell hybridomas. Hence, our data point out the expression of functional MHC class II molecules on human mast cells, reinforcing the implication of these cells in the defense mechanisms of acquired immunity.     

A role for HLA-DO as a co-chaperone of HLA-DM in peptide loading of MHC class II molecules

In B cells, the non-classical human leukocyte antigens HLA-DO (DO) and HLA-DM (DM) are residents of lysosome-like organelles where they form tight complexes. DM catalyzes the removal of invariant chain-derived CLIP peptides from classical major histocompatibility complex (MHC) class II molecules, chaperones them until peptides are available for loading, and functions as a peptide editor. Here we show that DO preferentially promotes loading of MHC class II molecules that are dependent on the chaperone activity of DM, and influences editing in a positive way for some peptides and negatively for others. In acidic compartments, DO is engaged in DR-DM-DO complexes whose physiological relevance is indicated by the observation that at lysosomal pH DM-DO stabilizes empty class II molecules more efficiently than DM alone. Moreover, expression of DO in a melanoma cell line favors loading of high-stability peptides. Thus, DO appears to act as a co-chaperone of DM, thereby controlling the quality of antigenic peptides to be presented on the cell surface.     

1,25-dihydroxycholecalciferol enhances the expression of MHC class II antigens and intercellular adhesion molecule-1 by human renal tubular epithelial cells

PURPOSE: Beside its role in calcium and phosphorus metabolism, 1,25-dihydroxycholecalciferol (1,25-D3) exerts multiple effects on cytokine and major histocompatibility complex (MHC) class II expression in monocytes and lymphocytes. In different renal diseases tubular epithelial cells express MHC class II molecules and cell adhesion molecules,, such as intracellular adhesion molecule-1 (ICAM-1). Therefore, modulation of MHC class II and ICAM-1 expression in renal tubular epithelial cells by 1,25-D3 may be relevant to lymphocyte adhesion to tubular epithelial cells and immune mediated renal injury. However, the expression of MHC class II antigens and cellular adhesion molecules by renal tubular epithelial cells in response to 1,25-D3 has not been investigated. MATERIALS AND METHODS: We generated human renal tubular epithelial cells and SV40 transfected tubular epithelial cells to investigate immune modulation of 1,25 on renal epithelial cells. Major histocompatibility complex class II molecules and ICAM-1 were detected by a specific enzyme linked immunoassay. RESULTS: We found a dose-dependent increase of both constitutive and induced MHC class II and ICAM-1 expression in tubular epithelial cells stimulated with 1,25-D3. Dose-dependent stimulation of MHC class II and ICAM-1 expression was not restricted to primary human renal tubular epithelial cells but was also detected in SV40 transfected cells. CONCLUSIONS: Expression of MHC class II and ICAM-1 is crucial for antigen presentation by and lymphocyte adhesion to renal tubular epithelial cells. Modulatory effects of 1,25-D3 on immune accessory function of renal tubular epithelial cells may be of clinical significance in renal diseases.     

Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells

Here, we show that bacteria induce de novo synthesis of both major histocompatability complex (MHC) class I and II molecules in a mouse dendritic cell culture system. The neo-biosynthesis of MHC class I molecules is delayed as compared with that of MHC class II. Furthermore, bacteria stabilize MHC class I molecules by a 3-fold increase of their half-life. This has important consequences for the capacity of dendritic cells to present bacterial antigens in the draining lymph nodes. In addition, a model antigen, ovalbumin, expressed on the surface of recombinant Streptococcus gordonii is processed and presented on MHC class I molecules. This presentation is 10(6) times more efficient than that of soluble OVA protein. This exogenous pathway of MHC class I presentation is transporter associated with antigen processing (TAP)-dependent, indicating that there is a transport from phagolysosome to cytosol in dendritic cells. Thus, bacteria are shown to be a potentially useful mean for the correct delivery of exogenous antigens to be presented efficiently on MHC class I molecules.     

Attenuation of HLA-DR expression by mononuclear phagocytes infected with Mycobacterium tuberculosis is related to intracellular sequestration of immature class II heterodimers

MHC class II expression was examined in macrophages infected with Mycobacterium tuberculosis. IFN-gamma increased the surface expression of class II molecules in THP-1 cells and this was markedly reduced in cells infected with M. tuberculosis. Despite this effect, steady state levels of HLA-DRalpha, HLA-DRbeta, and invariant (Ii) chains were equivalent in control and infected cells. Metabolic labeling combined with pulse-chase experiments and biochemical analysis showed that the majority of class II molecules in infected cells became resistant to endoglycosidase H, consistent with normal Golgi processing. However, results of intracellular staining and dual color confocal microscopy revealed a significant defect in transport of newly synthesized class II molecules through the endocytic compartment. Thus, compared with findings in control cells, class II molecules in infected cells colocalized to a minimal extent with a lysosomal-associated membrane protein-1+ endosomal compartment. In addition, in contrast to control cells, class II molecules in infected cells failed to colocalize with endocytosed BSA under conditions where this marker is known to label late endosomes, lysosomes, and the MHC class II compartment. Consistent with defective transport along the endocytic pathway, the maturation of SDS-stable class II alphabeta dimers--dependent upon removal of Ii chain and peptide loading of class II dimers in the MHC class II compartment--was markedly impaired in M. tuberculosis-infected cells. These findings indicate that defective transport and processing of class II molecules through the endosomal/lysosomal system is responsible for diminished cell surface expression of MHC class II molecules in cells infected with M. tuberculosis.     

MHC class II transport from lysosomal compartments to the cell surface is determined by stable peptide binding, but not by the cytosolic domains of the alpha- and beta-chains

Inside APCs, MHC class II molecules associate with antigenic peptides before reaching the cell surface. This association takes place in compartments of the endocytic pathway, more related to endosomes or lysosomes depending on the cell type. Here, we compared MHC class II transport from endosomal vs lysosomal compartments to the plasma membrane. We show that transport of MHC class II molecules to the cell surface does not depend on the cytosolic domains of the alpha- and beta-chains. In contrast, the stability of the alphabeta-peptide complexes determined the efficiency of transport to the cell surface from lysosomal, but not from endosomal, compartments. In murine B lymphoma cells, SDS-unstable and -stable complexes were transported to the cell surface at almost similar rates, whereas after lysosomal relocalization or in a cell line in which MHC class II molecules normally accumulate in lysosomal compartments, stable complexes were preferentially addressed to the cell surface. Our results suggest that when peptide loading occurs in lysosomal compartments, selective retention and lysosomal degradation of unstable dimers result in the expression of highly stable MHC class II-peptide complexes at the APC surface.     

The importance of the back-signal from T cells into antigen-presenting cells in determining susceptibility to parasites

It has long been known that certain MHC class II genes can dominantly suppress immune responses and so increase susceptibility to parasite infections, but the mechanism has been unclear. Recent work has revealed one way in which this form of suppression may operate, through gating by MHC class II molecules of the back-signal from activated T cells into macrophages. The two known suppressive genes of the mouse are expressed in macrophages more extensively than are other class II genes. This is associated with suppression of IL-4 production resulting, we infer, from overproduction in the macrophages of IL-12, the counter-cytokine to IL-4. The lack of IL-4 may itself be immunosuppressive, even for Th2 responses, and excess IL-12 can overinduce the antiproliferative cytokine IFN-gamma. Although this mechanism requires further substantiation, we believe that it offers a reasonable answer to an old conundrum.     

How HLA-DM affects the peptide repertoire bound to HLA-DR molecules

Considerable progress has been made in the field of major histocompatibility complex (MHC) class II-restricted antigen presentation. The analysis of mutant cell lines defective in antigen presentation revealed a central role for the nonclassical MHC class II molecule HLA-DM. Cell biological and biochemical characterization of HLA-DM provided deeper insight into the molecular mechanisms underlying the loading process: HLA-DM accumulates in acidic compartments, where it stabilizes classical class II molecules until a high-stability ligand occupies the class II peptide binding groove. Thus, HLA-DM prevents empty alpha beta dimers from functional inactivation at low endosomal/lysosomal pH in a chaperone-like fashion. In the presence of peptide ligands, HLA-DM acts as a catalyst for peptide loading by releasing CLIP, the residual invariant chain-derived fragment by which the invariant chain is associated with the class II molecules during transport from the endoplasmic reticulum to the loading compartments. Finally, there is accumulating evidence that HLA-DM functions as a peptide editor that removes low-stability ligands, thereby skewing the class II peptide repertoire toward high-stability alpha beta: peptide complexes presentable to T cells.     

HLA-DM stabilizes empty HLA-DR molecules in a chaperone-like fashion

HLA-DM (DM) is a non-classical major histocompatibility complex (MHC) class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (II)-derived CLIP peptides thereby rendering the peptide binding groove accessible for antigenic peptide loading. However, in situations of peptide scarcity the fate of these DM:DR complexes is not known. We could show that DR molecules incubated at lysosomal pH in the absence of peptide rapidly undergo functional inactivation and aggregation. In the presence of DM, however, empty DR molecules were shown to be stabilised and kept receptive for peptide loading, with the degree of the stabilising effect of DM varying for different DR alleles. In addition, in lysosomal compartments a considerable fraction of DM was found to be stably associated with empty DR alpha beta dimers thereby preserving their functionality. Upon encounter with antigenic peptide the DM-associated DR molecules could be rapidly loaded, whereupon they did no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone that rescues uncharged alpha beta dimers. In view of the suggested shortage of self-peptides in the loading compartment, empty class II molecules that are kept receptive for loading by the chaperone function of DM may enable the antigen processing system to respond promptly to the challenge by newly entering antigens.     

Anti-TNF antibody modulates cytokine and MHC expression in cardiac allografts

Tumor necrosis factor-alpha (TNF) is a multifunctional cytokine evoked in response to alloantigen stimulation and may be involved in lymphocyte activation, adhesion molecule expression, and regulation of MHC class II antigens. Anti-TNF treatment prolongs cardiac allograft survival. We investigated the role of anti-TNF in the regulation of MHC class II antigens and cytokine mRNA expression of TNF, interferon-gamma (IFN), IL-2, IL-4, and IL-10 in cardiac allografts to elucidate its immunological mechanism. These in vivo studies were conducted using a rat MHC mismatch Brown-Norway to Lewis (BN to LEW) heterotopic cardiac transplant model. In control untreated rats, allografts were rejected at 6.8 +/- 0.6 days. Allograft survival was significantly prolonged to 12.7 +/- 1.4 days with anti-TNF treatment. MHC class II expression, analyzed by indirect immunofluorescence cytometry, demonstrated that MHC class II-positive cells increased by 25% in spleens of untreated allografted rats compared to naive rats, while anti-TNF-treated allografted rats had a similar percentage of MHC II cells as naives. Further, naive, untransplanted rats and both anti-TNF and untreated, transplanted rats had heart and spleens harvested on Day 5 post-transplant. Cytokine mRNA expression was determined by semiquantitative RT-PCR. In heart and spleen cells from naives, TNF mRNA expression was undetectable or very weak. However, in rejecting allografts and spleen cells from untreated recipients, TNF expression was remarkably increased, while anti-TNF attenuated this TNF expression in both heart graft and spleen cells. Furthermore, IL-2, IL-10, and IFN expression were absent in naive hearts. However, in untreated allografts IL-2, IL-10, and IFN were strongly expressed, which was markedly decreased after anti-TNF treatment. Finally, IL-4 expression was found equally in naive hearts, untreated allografts, and anti-TNF-treated allografts. These results suggest that anti-TNF antibody treatment may not only neutralize TNF activity but also play a role in altering cytokine mRNA expression and MHC class II expression.