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Biomedical subjects

G Haraldsen

Publications and source records attributed to G Haraldsen.

At least 19 recordsLinked to original sources
PubMed2002

B cell attracting chemokine 1 (CXCL13) and its receptor CXCR5 are expressed in normal and aberrant gut associated lymphoid tissue.

BACKGROUND AND AIMS: In mice, the B lymphocyte chemoattractant (BLC) CXC chemokine ligand 13 (CXCL13) is sufficient to induce a series of events leading to the formation of organised lymphoid tissue. Its receptor, CXCR5, is required for normal development of secondary lymphoid tissue. However, the human counterpart, B cell attracting chemokine 1 (BCA-1) has only been detected in the stomach and appendix and not in other parts of normal or diseased gut. Hence to elucidate the potential role of this chemokine and its receptor in human gut associated lymphoid tissue (GALT), we analysed their expression in normal intestine and ulcerative colitis (UC). METHODS: Frozen sections of surgical specimens were studied by multicolour immunofluorescence staining, in situ mRNA hybridisation, and reverse transcription-polymerase chain reaction. RESULTS: BCA-1 mRNA was detected in all normal colonic and UC specimens. BCA-1 was produced and accumulated in relation to peripheral dendritic elements of lymphoid follicles in Peyer's patches and normal colon, as well as in irregular lymphoid aggregates in UC lesions. BCA-1 was partially associated with the traditional follicular dendritic cell phenotype but also with extracellular fibrils in GALT structures. CXCR5 protein was expressed by mantle zone B cells and appeared at a high level on scattered germinal centre T cells. CONCLUSIONS: BCA-1 and CXCR5 are expressed in normal GALT structures as well as in irregular lymphoid aggregates in UC. This strongly suggests that BCA-1 plays an important role not only in the formation of normal GALT but also in the generation of aberrant lymphoid tissue in inflammatory bowel disease.

Adult↗
PubMed2001

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

BACKGROUND: Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. METHODS: An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. RESULTS: E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. CONCLUSION: Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.

Animals↗
PubMed2001

The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment.

Lymphocyte homing to secondary lymphoid tissue is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial selectin-mediated tethering and rolling, firm adhesion of lymphocytes requires rapid upregulation of lymphocyte integrin adhesiveness. This step is mediated in part by the HEV-derived chemokine SLC (secondary lymphoid-tissue chemokine, or CCL21) that binds to the CC chemokine receptor (CCR)7 on lymphocytes. However, the CC chemokine ELC (Epstein-Barr virus-induced molecule 1 ligand chemokine, or CCL19) shares the same receptor, and ELC transcripts have been observed in the T cell areas of lymphoid organs. Here, we show that perivascular ELC is transcytosed to the luminal surfaces of HEVs and enables efficient T cell homing to lymph nodes. In situ hybridization on sections of human tonsil showed no ELC mRNA in HEVs, but immunostaining revealed ELC protein in cytoplasmic vesicles of HEV cells. Furthermore, ELC injected into the footpads of mice entered the draining lymph nodes and was presented by HEVs. Finally, intracutaneous injections of ELC in mice lacking functionally relevant ELC and SLC (plt/plt mice) restored T cell trafficking to draining lymph nodes as efficiently as SLC. We conclude that perivascular ELC is transcytosed to the luminal surfaces of HEVs and participates in CCR7-mediated triggering of lymphocyte arrest.

Animals↗
PubMed2000

Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: Epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.

The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4(+) and CD8(+) T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9(+), and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9(-). TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.

Animals↗
PubMed2000

Human intestinal endothelium shows high susceptibility to cytomegalovirus and altered expression of adhesion molecules after infection.

Human cytomegalovirus (HCMV) causes gastro intestinal disease with ulcerations, apparently as a consequence of cytopathic damage to endothelial cells (EC) and subsequent microvascular obliteration. In this study we showed that cultured human intestinal microvascular endothelial cells (HIMEC) are much more susceptible to HCMV infection than human umbilical vein endothelial cells (HUVEC). When both cell types were challenged with a clinical isolate of HCMV (10 pfu per cell), 30% of HIMEC expressed HCMV immediate early proteins, but only 10% of HUVEC. Enhanced susceptibility was also reflected in the expression of early and late HCMV proteins. In addition, the interleukin-1beta (IL-1beta)-induced cellular expression of adhesion molecules differed between HIMEC and HUVEC after HCMV-infection. E-selectin was unaffected in HUVEC but increased in HIMEC, whereas vascular cell adhesion molecule (VCAM)-1 was increased in HUVEC but decreased in HIMEC. Furthermore, HCMV-infection enhanced the expression of intercellular adhesion molecule (ICAM)-1 in both cell types. In conclusion, the enhanced susceptibility to HCMV infection observed in HIMEC and the elevated expression of E-selectin and ICAM-1 observed in these cells may provide an indication to the liability of developing gastrointestinal HCMV disease and may have a possible relevance to the survival of intestinal transplants.

Cell Adhesion Molecules↗
PubMed1999

The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.

Lymphocytes that are responsible for regional (tissue-specific) immunity home from the blood to the intestines, inflamed skin or other sites through a multistep process involving recognition of vascular endothelial cells and extravasation. Chemoattractant cytokine molecules known as chemokines regulate this lymphocyte traffic, in part by triggering arrest (stopping) of lymphocytes rolling on endothelium. Here we show that many systemic memory T cells in blood carry the chemokine receptor CCR4 and therefore respond to its ligands, the chemokines TARC and MDC. These cells include essentially all skin-homing cells expressing the cutaneous lymphocyte antigen and a subset of other systemic memory lymphocytes; however, intestinal (alpha4beta7+) memory and naive T cells respond poorly. Immunohistochemistry reveals anti-TARC reactivity of venules and infiltration of many CCR4+ lymphocytes in chronically inflamed skin, but not in the gastrointestinal lamina propria. Moreover, TARC induces integrin-dependent adhesion of skin (but not intestinal) memory T cells to the cell-adhesion molecule ICAM-1, and causes their rapid arrest under physiological flow. Our results suggest that CCR4 and TARC are important in the recognition of skin vasculature by circulating T cells and in directing lymphocytes that are involved in systemic as opposed to intestinal immunity to their target tissues.

Chemokine CCL17↗
PubMed1999

Heterogeneity of endothelial cells: the specialized phenotype of human high endothelial venules characterized by suppression subtractive hybridization.

High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs ( approximately 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH). Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.

Amino Acid Sequence↗
PubMed1999

Regional specialization in the mucosal immune system: primed cells do not always home along the same track.

According to the current paradigm of lymphocyte trafficking, primed B and T cells extravasate in the intestinal lamina propria chiefly by means of the mucosal homing receptor alpha4beta7, which interacts with the vascular addressin MAdCAM-1. However, as discussed here, this mechanism cannot explain the preferential homing of B cells with a high level of J-chain expression to mucosal effector sites outside the gut.

Animals↗
PubMed1999

Culture characterization of differentiated high endothelial venule cells from human tonsils.

High endothelial venules (HEV) are specialized vessels that support abundant lymphocyte emigration from peripheral blood into secondary lymphoid organs. HEV endothelial cells (HEVEC) exhibit particular structural and functional features, including secretion of the HEV-specific extracellular matrix protein hevin and an array of uniquely glycosylated counter-receptors for L-selectin expressed on lymphocytes. These ligands are collectively called the peripheral lymph node addressin (PNAd), originally defined by the monoclonal antibody MECA-79. PNAd expression was used to purify HEVEC by positive immunoselection from enzyme-digested human tonsils after negative immunoselection for other cells. Purified HEVEC maintained secretion of hevin and homogenous expression of intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2 (CD102), and CD31, at high levels following 8 days in culture. Expression of functional PNAd was maintained during the first 4 to 5 days of culture but decreased gradually and disappeared on day 8, while the expression of CD34 remained strong. However, the CD34 glycoform shifted toward the in situ phenotype of flat-walled vessels, suggesting that the observed loss of L-selectin binding determinants and MECA-79 antigen was due to down-regulation of the glycosyl- and sulfo-transferases essential for their expression. Our rapid and reproducible method to establish HEVEC cultures provides a useful mechanistic tool for identification of the factors that induce and maintain the HEV phenotype, as well as a source for isolation of HEV-specific genes.

Calcium-Binding Proteins↗
PubMed1998

Rapid secretion of prestored interleukin 8 from Weibel-Palade bodies of microvascular endothelial cells.

Interleukin (IL)-8, a C-X-C chemokine, activates integrin-mediated adhesion of neutrophils. Presentation of IL-8 on the endothelial cell surface may promote leukocyte extravasation. We found that cultured human microvascular endothelial cells from the intestine (HIMEC) and from nasal polyps (PMEC), but not human umbilical vein endothelial cells (HUVEC), contained IL-8 in intracellular granules that coexpressed von Willebrand factor (vWf ). This observation was corroborated by the immunohistochemical observation of double-positive granules (IL-8(+)vWf+) in vessels of small and large intestine, nasal mucosa, and skin, whereas umbilical cords revealed no endothelial IL-8. After treatment of HIMEC or PMEC with histamine or thrombin, a dramatic increase in supernatant IL-8 concentration was observed within 3 min, whereas no increase in IL-8 was detected in supernatants of identically treated HUVEC cultures. Histamine or thrombin treatment also caused IL-8-containing granules to rapidly disappear from HIMEC. In HUVEC, IL-8-containing granules were inducible by treatment with recombinant human IL-1beta for 24 h; additional histamine treatment doubled IL-8 secretion from HUVEC in the same rapid manner observed for mucosal EC. These data suggested that IL-8 prestored in microvascular endothelial cells may provide a rapid pathway for specific activation of neutrophil adhesion at sites of acute inflammation.

Cell Adhesion↗
PubMed1998

Major histocompatibility complex class II-dependent antigen presentation by human intestinal endothelial cells.

BACKGROUND & AIMS: In the normal gut, human intestinal microvascular endothelial cells (HIMECs) express major histocompatibility complex (MHC) class II molecules. Enhanced expression is found in chronic inflammation. We examined the cytokine regulation of MHC class II molecules and the associated invariant chain (Ii) in HIMECs and investigated whether such cells can process and present a complex protein antigen to T cells. METHODS: Enzyme-linked immunosorbent assay, flow cytometry, immunoelectron microscopy, as well as T-cell activation assay with HIMECs and HLA-DR-restricted T-cell clones were employed. RESULTS: In unstimulated HIMEC monolayers, HLA-DR, -DP, and -DQ and Ii were undetectable at the protein level, but interferon gamma (IFN-gamma) (100 U/mL) induced expression that peaked for DR after 2-3 days, for DP after 4-6 days, for DQ after 10-12 days, and for Ii after 2-3 days. Tumor necrosis factor alpha had no effect alone but enhanced class II expression in combination with IFN-gamma, most notably for DQ and DP. HLA-DR3-restricted and Mycobacterium tuberculosis heat shock 65-kilodalton-specific T-cell clones were activated to produce IFN-gamma in response to relevant antigen presented by IFN-gamma-treated HIMECs. This response was inhibited by blocking monoclonal antibody to HLA-DR and by chloroquine when compared to professional antigen-presenting cells, HIMECs activated T-cell clones quite efficiently. CONCLUSIONS: These data suggest that microvascular endothelial cells can present complex protein antigens in the human gut.

Antigen Presentation↗
PubMed1998

Cytokine profiles of cultured microvascular endothelial cells from the human intestine.

BACKGROUND AND AIMS: Cytokine production by endothelial cells, has, for practical reasons, been chiefly studied in human umbilical vein endothelial cells (HUVEC) but, because tissue-specific differences apparently exist, the role of human intestinal microvascular endothelial cells (HIMEC) as a source of mucosal cytokines was also assessed. METHODS: The expression of cytokine transcripts in HIMEC was screened by means of reverse transcription polymerase chain reaction (RT-PCR) and compared with cytokine profiles of HUVEC. Production of cytokines was investigated by bioassay and enzyme linked immunosorbent assay (ELISA). RESULTS: In the basal unstimulated state, HIMEC and HUVEC cultures contained detectable mRNA for interleukin (IL)-3, IL-7, IL-8, IL-11, IL-14, IL-15, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and granulocytemacrophage colony stimulating factor (GM-CSF). However, message was undetectable for IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p40, IL-13, and interferon (IFN)-gamma in the resting as well as the stimulated state. Stimulation of HIMEC and HUVEC with recombinant human (rh) IL-1 beta or rhTNF-alpha induced cell associated bioactive IL-1 alpha but not IL-1 beta, as well as enhanced secretion of both IL-6 and IL-8. Furthermore, transcript levels for GM-CSF and TNF-alpha were enhanced by rhIL-1 beta or rhTNF-alpha in both cell types. Supernatants from Th1-like or Th0-like gluten reactive intestinal T cell clones derived from patients with coeliac disease elicited cytokine profiles in both HIMEC and HUVEC similar to those revealed after rhIL-1 beta or rhTNF-alpha stimulation. CONCLUSIONS: These data demonstrate that the intestinal microvascular endothelium may contribute to the cytokine network of the intestinal mucosa with the ability to respond to locally generated cytokines and to produce potent inflammatory mediators.

Celiac Disease↗
PubMed1998

Cellular and molecular mechanisms for induction of mucosal immunity.

The epithelial glycoprotein called secretory component (SC) is quantitatively the most important receptor of the immune system because it is responsible for external transport of locally produced polymeric IgA (pIgA) to generate remarkably large amounts of secretory IgA. Antibodies of this type constitute the major mediators of specific humoral immunity. Transmembrane SC belongs to the Ig supergene family and functions as a common pIg receptor, also translocating pentameric IgM externally to form secretory IgM. The B cells responsible for mucosal production of Ig polymers are initially stimulated in organized mucosa-associated lympho-epithelial structures, particularly the Peyer's patches in the distal small intestine; from these inductive sites they migrate ("home") as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory effector site. There is currently great interest in exploiting this integrated or "common" mucosal immune system for oral vaccination against pathogenic infectious agents and also to induce tolerance in T cell-mediated auto-immune diseases. However, much remains to be learned about mechanisms for antigen uptake and processing necessary to elicit stimulatory or suppressive mucosal immune responses in humans. Moreover, evidence is emerging for the existence of considerable regionalization with regard to functional links between inductive sites and effector sites of mucosal immunity.

Animals↗
PubMed1997

Mechanism for enhanced external transfer of dimeric IgA over pentameric IgM: studies of diffusion, binding to the human polymeric Ig receptor, and epithelial transcytosis.

Transport of polymeric Igs (pIgA and pIgM) across secretory epithelia is mediated by the polymeric Ig receptor (pIgR), also known as the transmembrane secretory component. Compared with local production, external transfer of pIgA is favored 6- to 12-fold over that of pIgM on a molar basis. This transfer may be modulated at several levels: diffusion through matrix and basement membranes, ligand affinity for pIgR, and efficiency of epithelial transcytosis. To investigate these possibilities, we compared the ability of Madin-Darby canine kidney epithelial cells transfected with human pIgR to transport pIgA vs pIgM from the basolateral to the apical face, and examined the inhibitory effect of various filter types used for mounting of the monolayer. Binding data showed that pIgR bound pIgA and pIgM with similar affinity. Internalization of both ligands was fast and took place at similar rates; transcytosis was also found to be equally efficient at the molar level. Thus, the overall rate of transport across the epithelial monolayer was comparable for pIgA and pIgM, and was not further enhanced by ligand stimulation over a 20-fold increased concentration level. Conversely, pIgA had a considerable advantage over pIgM in passive diffusion assays performed in vitro. Moreover, in situ immunofluorescence staining showed retention of IgM over IgA and IgG in mucosal basement membrane zones, in contrast to the preferential epithelial uptake of IgA and, less so, IgM. The biologic consequences of the highly efficient epithelial pIg transport, and the diffusion advantage of pIgA over pIgM, are discussed in relation to the evolution and function of secretory Abs.

Animals↗
PubMed1997

Expression of functional VCAM-1 by cultured nasal polyp-derived microvascular endothelium.

Induction of endothelial vascular cell adhesion molecule-1 (VCAM-1) by interleukin (IL)-4 is believed to exert a major impact on the extravasation of leukocyte subsets in allergic disease. This notion has recently been challenged because cultured microvascular endothelial cells (ECs) derived from various organs are unable to express VCAM-1 after exposure to IL-4. In this study, we have established a method for isolation and culture of nasal polyp-derived microvascular ECs and report their cytokine-regulated VCAM-1 expression. With a combination of cell enzyme-linked immunosorbent assay, flow cytometry, and reverse transcription polymerase chain reaction, such expression was shown to be induced in a dose- and time-dependent manner not only by IL-1 beta and tumor necrosis factor-alpha but also by IL-4 and IL-13. Therefore, the response of nasal microvascular ECs did not harmonize with that of counterparts from several other tissues. IL-4 or IL-13 combined with submaximal concentrations of IL-1 beta or tumor necrosis factor-alpha increased VCAM-1 expression in a synergistic manner. VCAM-1 was functional as shown by antibody-mediated inhibition of leukocyte adhesion. Taken together, our results supported the notion that selective VCAM-1 induction by IL-4 and IL-13 plays an important role for the preferential recruitment of eosinophils and T lymphocytes seen in human airways affected by allergy.

Antigens, CD↗
PubMed1997

Mucosal immunity--a major adaptive defence mechanism.

The epithelial glycoprotein called secretory component (SC) is quantitatively the most important receptor of the immune system because it is responsible for external transport of locally produced polymeric IgA (pIgA) to generate remarkably large amounts of secretory IgA. Antibodies of this type constitute the major mediators of specific humoral immunity. Transmembrane SC belongs to the Ig supergene family and functions as a common pIg receptor, also translocating pentameric IgM externally to form secretory IgM. The B cells responsible for mucosal pIg production are initially stimulated in organized mucosa-associated lymphoepithelial structures, particularly the Peyer's patches in the distal small intestine; from these inductive site they migrate as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory effector site. There is currently great interest in exploiting this integrated or "common" mucosal immune system for oral vaccination against pathogenic infectious agents and also to induce tolerance in T cell-mediated autoimmune diseases. However, much remains to be learned about mechanisms for antigen uptake and processing necessary to elicit stimulatory or suppressive mucosal immune responses. Moreover, evidence is emerging for the existence of considerable regionalization with regard to functional links between inductive sites and effecter sites of mucosal immunity.

Animals↗