Trichuris suis soluble products induce Rab7b expression and limit TLR4 responses in human dendritic cells.

Inflammatory immune disorders such as inflammatory bowel disease and multiple sclerosis are major health problems. Currently, the intestinal whipworm Trichuris suis is being explored in clinical trials to reduce inflammation in these diseases; however, the mechanisms by which the parasite affects the host immune system are not known. Here we determined the effects of T. suis soluble products (SPs) on Toll-like receptor-4 (TLR4)-stimulated human dendritic cells (DCs) using Illumina bead chip gene arrays. Pathway analysis of lipopolysaccharide-stimulated DCs with or without T. suis treatment showed that co-stimulation with T. suis SPs resulted in a downregulation of both the myeloid differentiation primary response gene 88-dependent and the TIR-domain-containing adaptor-inducing interferon-ß-dependent signalling pathways triggered by TLR4. These data were verified using quantitative real-time PCR of several key genes within these pathways and/or defining their protein levels. In addition, T. suis SPs induce Rab7b, a negative regulator of TLR4 signalling that interferes with its trafficking, which coincided with a reduced surface expression of TLR4. These data indicate that the mechanism by which T. suis SPs reduce inflammatory responses is through suppression of both TLR4 signalling and surface expression on DCs.

HLA-DRB1*03:01 and HLA-DRB1*04:01 modify the presentation and outcome in autoimmune hepatitis type-1.

The classical human leukocyte antigen (HLA)-DRB1*03:01 and HLA-DRB1*04:01 alleles are established autoimmune hepatitis (AIH) risk alleles. To study the immune-modifying effect of these alleles, we imputed the genotypes from genome-wide association data in 649 Dutch AIH type-1 patients. We therefore compared the international AIH group (IAIHG) diagnostic scores as well as the underlying clinical characteristics between patients positive and negative for these HLA alleles. Seventy-five percent of the AIH patients were HLA-DRB1*03:01/HLA-DRB1*04:01 positive. HLA-DRB1*03:01/HLA-DRB1*04:01-positive patients had a higher median IAIHG score than HLA-DRB1*03:01/HLA-DRB1*04:01-negative patients (P<0.001). We did not observe associations between HLA alleles and alanine transaminase levels (HLA-DRB1*03:01: P=0.2; HLA-DRB1*04:01; P=0.5); however, HLA-DRB1*03:01 was independently associated with higher immunoglobulin G levels (P=0.04). The HLA-DRB1*04:01 allele was independently associated with presentation at older age (P=0.03) and a female predominance (P=0.04). HLA-DRB1*03:01-positive patients received immunosuppressive medication and liver transplantation. In conclusion, the HLA-DRB1*03:01 and HLA-DRB1*04:01 alleles are both independently associated with the aggregate diagnostic IAIHG score in type-1 AIH patients, but are not essential for AIH development. HLA-DRB1*03:01 is the strongest genetic modifier of disease severity in AIH.

Cytotoxic T lymphocyte antigen-4 +49A/G polymorphism does not affect susceptibility to autoimmune hepatitis.

BACKGROUND & AIMS: Single nucleotide polymorphisms (SNP) in the Cytotoxic T lymphocyte antigen-4 gene (CTLA-4) have been associated with several autoimmune diseases including autoimmune Hepatitis (AIH). In this chronic idiopathic inflammatory liver disease, conflicting results have been reported on the association with a SNP at position +49 in the CTLA-4 gene in small patient cohorts. Here, we established the role of this SNP in a sufficiently large cohort of AIH patients. METHODS: The study population consisted of 672 AIH patients derived from academic and regional hospitals in the Netherlands and was compared with 500 controls selected from the 'Genome of the Netherlands' project cohort. Genotype frequencies were assessed by PCR for patients and by whole genome sequencing for controls. RESULTS: No significant differences in allele frequencies were found between patients and controls (G Allele: 40% vs 39%, P = 0.7). Similarly, no significant differences in genotype frequencies between patients and controls were found. Finally, there was no relation between disease activity and the G allele or AG and GG genotypes. CONCLUSION: The Cytotoxic T Lymphocyte Antigen-4 +49 A/G polymorphism does not represent a major susceptibility risk allele for AIH in Caucasians and is not associated with disease severity at presentation.

Genetic variations in interleukin-12 related genes in immune-mediated diseases.

The interleukin-12 (IL-12) family comprises a group of heterodimeric cytokines and their respective receptors that play key roles in immune responses. A growing number of autoimmune diseases has been found to be associated with genetic variation in these genes. Based on their respective associations with the IL-12 genes, autoimmune diseases appear to cluster in two groups that either show strong associations with the Th1/Th17 pathway (as indicated by genetic association with IL12B and IL23R) or the Th1/IL-35 pathway as the consequence of their association with polymorphisms in the IL12A gene region. The genetic associations are described in relation to what is known of the functionality of these genes in the various diseases. Comparing association data for gene families in different diseases may lead to better insight in the function of the genes in the onset and course of the disease.

Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody.

An mAb, NLDC-145, is described that specifically reacts with a group of nonlymphoid dendritic cells including Langerhans cells (LC), veiled cells (VC), and interdigitating cells (IDC). The antibody does not react with precursor cells in bone marrow and blood. Macrophages are not stained by the antibody, but a subpopulation of Ia+ peritoneal exudate cells is recognized. Possible relationships of the various nonlymphoid dendritic cell (NLDC) types are discussed.

Alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice.

A single intracheal dose of liposome-encapsuled dichloro-methylene-diphosphonate resulted in the elimination of alveolar macrophages (AM) from the lung, creating a model to study the in vivo role of AM in the pulmonary immune response. Using intratracheally administered trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), the kinetics of the response, the location and number of TNP-specific antibody-forming cells, and the different Ig classes of the antibodies produced were studied in AM-depleted animals. The results show that AM elimination has a dramatic effect on the pulmonary immune responses against TNP-KLH. An increase in APC in lung-associated lymph nodes and a prolongation of the response is found, as well as an introduction of APC in lung tissue. In both experimental groups, the majority of the TNP-specific antibodies produced was IgG, followed by IgA and IgE, while very few IgM antibodies could be detected. We conclude from these results that AM are likely to play a role in controlling the pulmonary immune response in a suppressive way, thereby limiting the possible damage caused by severe immune responses in lung tissue.

Identification of a soluble form of a ligand for the lymphocyte homing receptor.

Lymphocytes are engaged in constant trafficking from the blood into secondary lymphoid tissues, such as peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP). The initial step in this process is the binding of lymphocytes to high endothelial venules (HEV), and in the case of trafficking of cells to the PLN, it is required that they bear the L-selectin surface receptor. Using a chimeric protein, combining the extracellular domains of L-selectin with a human immunoglobulin (Ig) G1 Fc region (L-selectin-IgG), we have probed the expression of ligands for this receptor on HEV and in cell lysates. Two sulfated glycoproteins of 50 and 90 kD have been identified in lysates from PLN and MLN, but not PP. Here we show that the 50-kD molecule is secreted in organ cultures in vitro and is present in the blood of normal animals. Indeed, normal serum inhibits lymphocyte binding to HEV by approximately 50% in an in vitro assay. This inhibitory activity can be removed by passage of the serum over an L-selectin-IgG column and has a molecular mass of approximately 50 kD. We speculate on the possible reasons for secretion of a homing receptor ligand.

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.

Sialoadhesin on macrophages: its identification as a lymphocyte adhesion molecule.

In this study we present evidence that the mouse and rat sialoadhesin (originally named sheep erythrocyte receptor) on macrophages can function as a lymphocyte adhesion molecule. Lymphocytes were shown to bind to the splenic marginal zone, and lymph node subcapsular sinus and medulla in a frozen section assay. Selective depletion experiments showed that binding was mediated by macrophages. Adhesion was blocked by preincubation of the sections with monoclonal antibodies against mouse or rat sialoadhesin. Binding was temperature dependent, divalent cation independent, and involved sialic acid residues on the lymphocyte, as it could be inhibited by prior neuraminidase treatment or addition of the ganglioside GD1a. Binding to sialoadhesin was confirmed using the purified receptor and was observed among T cells, T blasts, B cells, and B blasts. Isolated macrophages or dendritic cells showed little binding. Sialoadhesin provides the first example of a macrophage-restricted lymphocyte adhesion molecule.

The influence of afferent lymphatic vessel interruption on vascular addressin expression.

Tissue-selective lymphocyte homing is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. These vessels, the post capillary high endothelial venules (HEV), are found in organized lymphoid tissues, and at sites of chronic inflammation. Lymphocytes bearing specific receptors, called homing receptors, recognize and adhere to their putative ligands on high endothelial cells, the vascular addressins. After adhesion, lymphocytes enter organized lymphoid tissues by migrating through the endothelial cell wall. Cells and/or soluble factors arriving in lymph nodes by way of the afferent lymph supply have been implicated in the maintenance of HEV morphology and efficient lymphocyte homing. In the study reported here, we assessed the influence of afferent lymphatic vessel interruption on lymph node composition, organization of cellular elements; and on expression of vascular addressins. At 1 wk after occlusion of afferent lymphatic vessels, HEV became flat walled and expression of the peripheral lymph node addressin disappeared from the luminal aspect of most vessels, while being retained on the abluminal side. In addition, an HEV-specific differentiation marker, defined by mAb MECA-325, was undetectable at 7-d postocclusion. In vivo homing studies revealed that these modified vessels support minimal lymphocyte traffic from the blood. After occlusion, we observed dramatic changes in lymphocyte populations and at 7-d postsurgery, lymph nodes were populated predominantly by cells lacking the peripheral lymph node homing receptor LECAM-1. In addition, effects on nonlymphoid cells were observed: subcapsular sinus macrophages, defined by mAb MOMA-1, disappeared; and interdigitating dendritic cells, defined by mAb NLDC-145, were dramatically reduced. These data reveal that functioning afferent lymphatics are centrally involved in maintaining normal lymph node homeostasis.

The effect of NOD2 activation on TLR2-mediated cytokine responses is dependent on activation dose and NOD2 genotype.

The mechanism by which mutations in NOD2 predispose to Crohn's disease (CD) is incompletely understood. In mice, NOD2 has been found to function as a negative regulator of Toll-like receptor 2 (TLR2) signaling. In contrast, studies in humans so far showed no negative regulatory interaction between NOD2 and TLR2, and in fact suggest a synergistic effect between the two. Here, we show that this interaction is dose dependent. Adding low doses of muramyl dipeptide (MDP) to TLR2 primed monocytes results in a significant increase in cytokine production, whereas adding higher doses of MDP led to a striking downregulation of the responses. This downregulation by high-dose MDP does not occur in monocytes from NOD2-deficient patients. The inhibitory role of NOD2 at high concentrations of MDP implicates a safety mechanism to prevent exaggerated antibacterial immune responses in the gut to high or perpetuating bacterial load. This regulatory mechanism is lost in NOD2-deficient CD patients.

Mice bearing the E mu-myc and E mu-pim-1 transgenes develop pre-B-cell leukemia prenatally.

Previously, it has been shown that E mu-pim-1 transgenic mice are predisposed to T-cell lymphomas, whereas E mu-myc transgenic mice are predisposed to pre-B-cell lymphomas. Here we show that double-transgenic E mu-myc E mu-pim-1 mice exhibit pre-B-cell leukemia in utero. Upon transplantation into recipient mice, embryo-derived double-transgenic leukemic cells frequently progressed to highly malignant monoclonal tumors, indicating that additional (epi)genetic events had occurred during the progression of the disease.

Cells in the marginal zone of the spleen.

The marginal zone of the spleen forms an intriguing area in which a variety of cell types are combined. Several of these cell types seem to have a fixed position in the marginal zone, such as the marginal zone macrophages, the marginal metallophilic macrophages at the inner border, and, to a lesser extent, the marginal zone B cells. For other cell types--T lymphocytes, small B cells, and dendritic cells--the marginal zone is only a temporary residence. It is this combination of relatively sessile cell populations and the continuous influx and passing of bloodborne immunocompetent cells that turn the marginal zone into a dynamic area, particularly apt for antigen processing and recognition. In no other lymphoid organ can such a unique combination of cells and functions be found. The opening of the arterial blood stream in the marginal sinuses results in a reduction of the velocity of the blood stream, and antigens are initially screened in the marginal zone. To this, extremely potent phagocytic cells, the marginal zone macrophages, are present which can take up and phagocytize large foreign particles, such as bacteria and effete red blood cells. Further filtration of the blood takes place in the filtration beds of the red pulp. The marginal zone macrophages express membrane receptors for bacterial polysaccharides which lead to efficient phagocytosis, probably even in the absence of prior opsonization. Antigenic fragments produced this way can be taken up by dendritic cells that enter the spleen by the blood as part of a mobile surveillance immune system. Dendritic cells present antigen to T cells in the outer area of the T cell-dependent PALS, leading to clustering and enrichment of antigen-specific T cells. Antigens in the marginal zone can also directly associate with memory B cells thought to reside here for longer times, having intimate contact with the marginal zone macrophages. B memory cells then migrate into the PALS and present antigen to T cells. The marginal zone therefore functions not only as an area of initial filtration and phagocytosis of antigens from the blood, but also as a site of lymphocyte emigration. Some of the incoming T and B lymphocytes in the recirculating pool enter the white pulp from the marginal zone. The underlying force and selective molecular mechanisms that guide this migration are unknown. Both B and T lymphocytes recirculate through the outer PALS area on their way to the follicles and the inner PALS, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Macrophage subset repopulation in the spleen: differential kinetics after liposome-mediated elimination.

Different macrophage subsets can be discriminated in the well defined compartments of the mouse spleen by specialized functions and the presence of specific surface determinants. Red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages are eliminated simultaneously within 24 hr by a single injection with liposome-entrapped dichloromethylene diphosphonate (DMDP). After such elimination, these subsets show a striking difference in their kinetics of reappearance: Red pulp macrophages are back in normal numbers after 1 week, the marginal metallophilic macrophages take 2 weeks to regain fully their position at the border of the marginal zone and periarteriolar lymphocyte sheath, but it takes over 1 month for complete reappearance of the marginal zone macrophages. Marginal zone lymphocytes, also affected by treatment with the liposome-entrapped drug, reappeared in the marginal zone within 2 weeks, indicating that marginal zone macrophages are not required for their localization and/or retention there. Approximately 2 weeks after treatment, all cells in the spleen have returned to normal numbers with the exception of marginal zone macrophages, which can be found only sporadically at that time. The results indicate that these macrophage subpopulations must have different precursor requirements. The differential reappearance of the macrophages creates the possibility of studying lineage analysis and will help to unravel the precise function of the marginal zone macrophages and marginal metallophilic macrophages in particular.

Repopulation of macrophages in popliteal lymph nodes of mice after liposome-mediated depletion.

Macrophages lining the subcapsular sinus (SCS) and those located in the medulla of popliteal lymph nodes (PLN) of mice were eliminated after subcutaneous (s.c.) injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes. No effect of liposome-entrapped Cl2MDP could be seen on nonphagocytic cells, e.g., interdigitating cells (IDC) and B- and T-lymphocytes. One month after injection the eliminated subsets of macrophages were still absent. After 2 mo a small number of macrophages had reappeared along the SCS and in the medulla of the PLN of a few animals. Complete repopulation of the PLN with macrophages was observed only after 5 mo. This extremely long repopulation time could be shortened drastically by local administration of Freund's complete adjuvant (FCA). A small number of macrophages reappeared along the SCS and in the medulla 1 wk after FCA and after 3 wk the repopulation of the PLN with macrophages was complete. Such rapid repopulation of macrophages was not achieved after s.c. injection of paraffin oil or paratyphoid vaccine. These results indicate that the normal rate of influx of mononuclear phagocytes into the PLN is low, but that it can be sped up after administration of FCA.

Macrophage phenotype regulation by colony-stimulating factors at bone marrow level.

Macrophages (m phi s) can be divided into several subpopulations, which differ in phenotype, function, and localization patterns. However, little is known about the mechanisms that regulate this heterogeneity. We investigated whether m phi heterogeneity is regulated by colony-stimulating factors (CSFs) at the bone marrow level. By clonal expansion of bone marrow-derived precursor cells in the presence of CSF-1, granulocyte-macrophage CSF or multi-CSF (interleukin-3), phenotypic heterogeneity was observed between m phi colonies. Heterogeneity was found especially when different CSF culture conditions were used but also between m phi colonies derived under the same CSF culture condition. Our results illustrate that CSFs from the bone marrow hemopoietic microenvironment are important for the induction of phenotypic heterogeneity within the progeny of cloned m phi precursor cells during maturation and differentiation in vitro.

Establishment of bone marrow-derived macrophage cell lines by soft-agar cloning: regulation of macrophage heterogeneity at bone marrow level.

To study the regulation of macrophage (m phi) heterogeneity at bone marrow level, we developed a liquid culture system in which bone marrow-derived soft-agar colonies were expanded in the presence of colony-stimulating factor-1 (CSF-1). Several cloned m phi precursor cells were established as CSF-1-dependent cell lines, and were analyzed for morphological, phenotypic and functional characteristics. The continuously proliferating cell lines expressed both immature and mature m phi markers. Only minor differences between the established cell lines were detected. Thus, our results show that during long-term culture CSF-1-responsive cloned m phi precursor cells develop along identical pathways, giving rise to progeny with comparable phenotype and function in vitro.

Antigen-bearing Langerhans cells in skin draining lymph nodes: phenotype and kinetics of migration.

Application of the fluorescent contact sensitizer Rhodamin B on mouse epidermis was used to study the migration kinetics of Langerhans cells into the draining lymph nodes. The expression of the dendritic cell markers NLDC-145 and MIDC-8 was followed over time to determine the correlation between these markers and Langerhans cell migration. In contrast with its high expression on intraepidermal Langerhans cells, the expression of NLDC-145 on dendritic cells in the draining lymph node was low at 24 h but increased at later times; in contrast, MIDC-8 expression on dendritic cells decreased. Ten days after Rhodamin B application, antigen-bearing Langerhans cells were still present in the epidermis; application of another unrelated contact sensitizer to the epidermis at this time did not lead to migration of these residual Langerhans cells. These results indicate that not all antigen-bearing Langerhans cells migrate from the skin after application of a contact sensitizer, indicating that signals in addition to simple antigen binding are necessary for migration. During this migration from epidermis to lymph nodes Langerhans cells undergo phenotypic changes. The decreased expression of the endosomal antigens MIDC-8 and MOMA-2 correlates with differentiation from predominantly antigen-processing cells to predominantly antigen-presenting cells. The reduced expression of NLDC-145 is discussed in light of a Langerhans cell-independent pathway of antigen transportation from skin to lymph node.

Non-sensitizing epicutaneous skin tests prevent subsequent induction of immune tolerance.

Oral administration of nickel or chromium to naive guinea pigs results in immune unresponsiveness to subsequent induction of allergic contact hypersensitivity. Such "oral tolerance" depends on the oral dose, is antigen specific, T-suppressor-cell mediated, and very persistent. In contrast, oral antigen administration to sensitized animals results at best in transient desensitization. Here we report that even non-sensitizing epicutaneous skin contacts prevented the subsequent induction of oral tolerance. These data support the view that primed T cells are less sensitive to suppressor T-cell function than naive T cells.

Effect of aging on epidermal dendritic cell populations in C57BL/6J mice.

The density and function of epidermal dendritic cell populations were investigated in aged C57BL/6J mice. The densities of both Langerhans cells (LC) and Thy-1+ dendritic epidermal cells were found to decrease with age. Epidermal cell suspensions from aged mice showed impaired immunologic function as assessed in vitro by the skin-lymphocyte reaction assay and by measuring the ability of epidermal cell suspensions to stimulate the proliferation of sensitized T cells in the presence of the sensitizing antigen. However, the capacity of LC to transport antigen from the skin to the draining lymph nodes was found in vivo to be comparable to that of young mice. Results of transplantation of bone marrow cells from young and old donors into irradiated recipients indicate that the decreased Langerhans cell density found in old mice may result from a deficiency in Langerhans cell bone marrow progenitors.