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Biomedical subjects

A W Thomson

Publications and source records attributed to A W Thomson.

At least 127 records · Page 7Linked to original sources
PubMed1995

Mode of action of tacrolimus (FK506): molecular and cellular mechanisms.

Tacrolimus, formerly known as FK506, is a macrolide antibiotic with immunosuppressive properties. Although structurally unrelated to cyclosporin A (CsA), its mode of action is similar. It exerts its effects principally through impairment of gene expression in target cells. Tacrolimus bonds to an immunophilin, FK506 binding protein (FKBP). This complex inhibits calcineurin phosphatase. The drug inhibits calcium-dependent events, such as interleukin-2 gene transcription, nitric oxide synthase activation, cell degranulation, and apoptosis. Tacrolimus also potentiates the actions of glucocorticoids and progesterone by binding to FKBPs contained within the hormone receptor complex, preventing degradation. The agent may enhance expression of the transforming growth factor beta-1 gene in a fashion analogous to that demonstrated for CsA. T cell proliferation in response to ligation of the T cell receptor is inhibited by tacrolimus. Type 1 T helper cells appear to be preferentially suppressed compared with type 2 T helper cells. T cell-mediated cytotoxicity is impaired. B cell growth and antibody production are affected indirectly by the suppression of T cell-derived growth factors necessary for these functions. Antigen presentation appears to be spared. The molecular events affected by tacrolimus continue to be discovered.

Gene Expression Regulation↗
PubMed1995

Combined effects of FK506 (tacrolimus) and cyclophosphamide on atypical B220+ T cells, cytokine gene expression and disease activity in MRL/MpJ-lpr/lpr mice.

Groups of female MRL/MpJ-lpr/lpr mice received either saline or FK506 (tacrolimus; 2 mg/kg intraperitoneally) three times weekly, cyclophosphamide (CY; 20 mg/kg) once monthly, or both drugs from 8 weeks of age. Median survival for untreated and CY-treated mice was 26 weeks, and for FK506- and FK506 + CY-treated groups was > or = 44 weeks. Severity of skin lesions and lymph node hyperplasia was markedly reduced by the drug combination, whereas either drug alone was less effective. FK506 or CY alone delayed the onset of proteinuria, but by 24 weeks all of these animals were positive. In contrast, drug combination reduced the prevalence of proteinuria to < or = 60% throughout the 44 weeks of study. Sequential monitoring of peripheral blood lymphocytes revealed that combination therapy but not monotherapy markedly reduced the proportion of atypical CD3+ B220+ and CD3+CD4-CD8- T cells. Neither FK506 nor CY affected the reduction in IL-2 and IL-4 mRNA levels observed in lymph nodes of diseased animals compared with normals. Although the drug combination also did not affect IL-2 mRNA levels, IL-4 mRNA transcripts were increased six-fold compared with saline-treated controls. IL-10 and interferon-gamma (IFN-gamma) mRNAs were induced by FK506, CY and by the drug combination. Serum levels of anti-dsDNA antibodies were reduced in all treatment groups. These data demonstrate improved efficacy of combined T and B cell-directed immunosuppression in murine lupus, associated with marked inhibition of atypical T cells and selective augmentation of IL-4 within the affected lymphoid tissue.

Animals↗
PubMed1995

IL-8/IL-8 receptor expression in psoriasis and the response to systemic tacrolimus (FK506) therapy.

Recently, the keratinocyte IL-8/IL-8 receptor (IL-8R) pathway has been implicated in the pathogenesis of psoriasis, and there is evidence that the potent macrolide immune suppressant tacrolimus (formerly FK506) can inhibit this pathway in vitro. In this study, determination of the expression of cytokine mRNAs in lesional skin of patients with active disease by reverse transcriptase polymerase chain reaction revealed transcripts for IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-8, IL-8R, IL-10, interferon-gamma (IFN-gamma), IL-2R and transforming growth factor-beta (TGF-beta), but not IL-2 or IL-4. IL-8 was the only cytokine expressed in affected skin of all patients but not in clinically normal skin of healthy subjects. In seven CD4+ T cell clones propagated from the lesional skin of an untreated psoriasis patient, IL-8 was expressed by the skin-derived T lymphocytes and not by feeder cells (irradiated autologous blood lymphocytes); IL-1 beta, IL-2, IL-6 and IL-10 were also expressed by some or all of the T cell clones. IL-8 mRNA was not detected in the skin of any patient after the start of systemic tacrolimus therapy; IL-1 beta, IL-6 and IFN-gamma transcripts were also reduced. By 12 weeks, the mean psoriasis area and severity index (PASI) had decreased from 18.8 to 3.8, a reduction of 80%. In the same post-treatment biopsies, however, message for IL-8R persisted. Estimation of circulating IL-8 levels by enzyme immunoassay showed that all patients with detectable IL-8 before treatment had decreased levels in response to treatment with tacrolimus; reductions in PASI scores were accompanied by decreases in IL-8 levels, that varied both in rate and extent. Partial relapse, which in a minority of patients followed the initial period of remission, and was precipitated by drug dose reduction, was accompanied by an increase in circulating IL-8. These findings add credence to the view that the IL-8/IL-8R autocrine/paracrine pathway may be important in the pathogenesis of psoriasis. They further suggest that interference with IL-8 production and/or that of other key chemokines may be an important mechanism underlying the therapeutic efficacy of tacrolimus, and other agents such as cyclosporin A, with similar molecular actions.

Adolescent↗
PubMed1995

Presensitization by skin grafting from major histocompatibility complex class I or major histocompatibility complex class II deficient mice identifies class I antigens as inducers of allosensitization.

Livers but not hearts are accepted spontaneously without immunosuppression when transplanted from B10 (KbAbEbDb) to C3H (KkAkEkDk) mice. Both organs however, undergo accelerated rejection in C3H recipients presensitized with B10 skin grafts. In this study, we have investigated further the role of functional cell-surface major histocompatibility complex (MHC class I or class II molecules in allosensitization. Skin from transgenic MHC class I (b2mmlUncbcr; AbEb) or class II (C2DTM, KbDb) gene 'knockout' mice was grafted onto naive recipients 2-3 weeks prior to whole organ transplantation. When C3H hosts were presensitized with skin from C2DTM (class II deficient) mice, they promptly rejected (within 4 days) subsequently transplanted B10 liver or heart allografts. In contrast, presensitization with skin from b2m (beta 2-m mutant; class I deficient) mice did not significantly affect the survival of either organ graft. Maximal sensitization was established by day 14 after skin grafting and persisted for at least 12 weeks. Splenocytes obtained from C3H mice sensitized with skin from B10, B6 (KbAbEbDb), or C2DTM but not from b2m mice exhibited an H-2b-specific cytolytic response when tested in cell-mediated lymphocytotoxicity assays. Sera from C3H mice sensitized with B10 or b2m skin contained high titres of cytotoxic activity specifically against H-2b class I. Taken together, these observations suggest that in the strain combination studied, MHC class I rather than class II molecules play an important role in allosensitization. The results indicate the potential importance of avoiding transplantation of organs into recipients of secondary grafts from donors that share human leucocyte antigen (HLA) class I antigens with the first donor.

Animals↗
PubMed1995

Generation of DC from mouse spleen cell cultures in response to GM-CSF: immunophenotypic and functional analyses.

In all tissues that have been studied to date, dendritic leucocytes constitute only a small proportion of total cells and are difficult both to isolate and purify. This study reports on a method for the propagation of large numbers of dendritic cells (DC) from mouse spleen using granulocyte-macrophage colony-stimulating factor (GM-CSF) and their characteristics. Within a few days of liquid culture in GM-CSF, B10 BR (H-2k, I-E+) mouse splenocytes formed loosely adherent myeloid cell clusters. Mononuclear progeny released from these clusters at and beyond 4 days exhibited distinct dendritic morphology and strongly expressed leucocyte common antigen (CD45), CD11b, heat-stable antigen, Pgp-1 (CD44) and intercellular adhesion molecule-1 (ICAM-1; CD54). The intensity of expression of the DC-restricted markers NLDC 145 and 33D1, the macrophage marker F4/80, and Fc gamma RII (CDw32) was low to moderate, whereas the cells were negative for CD3, CD45RA and NK1.1. High and moderate levels, respectively, of cell surface staining for major histocompatibility complex (MHC) class II (I-Ek) and the B7 antigens (counter-receptors of CTLA4, a structural homologue of CD28) were associated with potent stimulation of unprimed, allogeneic T cells (B10; H-2b, I-E-). DC propagated in a similar fashion from DBA/2 mouse spleen proved to be strong antigen-presenting cells (APC) for MHC-restricted, syngeneic T-helper type 2 (Th2) cell clones specifically responsive to sperm whale myoglobin. Footpad or intravenous injection of GM-CSF-stimulated B10.BR spleen-derived DC into B10 (H-2b, I-E-) recipients resulted in homing of the allogeneic cells to T-cell-dependent areas of lymph nodes and spleen, where they strongly expressed donor MHC class II antigen 1-2 days later. These findings indicate that cells can be propagated from fresh splenocyte suspensions that exhibit distinctive features of DC, namely morphology, motility, cell-surface phenotype, potent allogeneic and syngeneic APC function and in vivo homing ability. Propagation of DC in this manner from progenitors present in lymphoid tissue provides an alternative and relatively convenient source of high numbers of these otherwise difficult to isolate but functionally important APC.

Animals↗
PubMed1994

Isolation, phenotype, and allostimulatory activity of mouse liver dendritic cells.

Donor liver-derived dendritic cells (DC) have recently been identified within various lymphoid and nonlymphoid tissues of organ allograft recipients, including nonimmunosuppressed mice transplanted with and permanently accepting major histocompatibility complex (MHC)-disparate hepatic allografts. These findings have raised questions about the basis of the tolerogenicity of the liver--and, in particular, about the properties of liver-derived DC. To study further the structure, immunophenotype and allostimulatory activity of leukocytes resident in normal mouse (B10.BR;H-2k, I-Ek) liver, a procedure was developed to maximize the yield of viable, nonparenchymal cells (NPC) obtained following collagenase digestion of perfused liver fragments and density centrifugation (Percoll). These cells comprised populations expressing lymphoid and myeloid cell surface antigens. As compared with spleen cells, they proved good allostimulators of naive (B10; H-2b, I-E-) splenic T cells when tested in primary mixed leukocyte reactions (MLR). After overnight (18-hr) incubation of the NPC, enrichment for transiently adherent, low-density (LD) cells on metrizamide gradients permitted the recovery of low numbers of cells (approx. 2-5 x 10(5) per liver), many of which displayed distinct DC morphology. Flow cytometric analysis revealed that these cells were CD3-, CD4-, CD8-, and B220-, but strongly expressed CD45 (leukocyte-common antigen), and mild-to-moderate levels of CD11b, heat-stable antigen, and CD44. The cells also expressed moderate intensity of NLDC 145 but not 33D1, DC restricted markers which have been shown to be differentially expressed on mouse DC isolated from various organs. This DC-enriched population was more strongly MHC class II(I-Ek)+ than NPC, as determined by immunocytochemistry and flow cytometry and exhibited much more potent allostimulatory activity for naive T cells. These findings demonstrate that freshly isolated murine liver NPC, and perhaps their counterparts in situ, exhibit allostimulatory activity that is enhanced in the non-adherent, low-density (DC-enriched) fraction after overnight culture. They further suggest that the maturation of liver DC may play a key role in determining the immunogenicity and or tolerogenicity of hepatic allografts.

Animals↗
PubMed1994

Propagation of dendritic cell progenitors from normal mouse liver using granulocyte/macrophage colony-stimulating factor and their maturational development in the presence of type-1 collagen.

Within 1 wk of liquid culture in granulocyte/macrophage colony-stimulating factor (GM-CSF), normal B10 BR (H-2k I-E+) mouse liver nonparenchymal cells (NPC) formed loosely adherent myeloid cell clusters that have been shown to contain dendritic cell (DC) progenitors in similar studies of mouse blood or bone marrow. Mononuclear cell progeny released from these clusters at and beyond 4 d exhibited distinct dendritic morphology and were actively phagocytic. After 6-10 d of culture, these cells strongly expressed CD45, CD11b, heat stable antigen, and CD44. However, the intensity of expression of the DC-restricted markers NLDC 145, 33D1, and N418, and the macrophage marker F4/80, intercellular adhesion molecule 1, and Fc gamma RII was low to moderate, whereas the cells were negative for CD3, CD45RA, and NK1.1. Splenocytes prepared in the same way also had a similar range and intensity of expression of these immunophenotypic markers. Unlike the splenic DC, however, most of the GM-CSF-propagated putative liver DC harvested at 6-10 d expressed only a low level of major histocompatibility complex (MHC) class II (I-Ek), and they failed to induce primary allogeneic responses in naive T cells, even when propagated additionally in GM-CSF and tumor necrosis alpha and/or interferon gamma-supplemented medium. However, when 7-d cultured GM-CSF-stimulated liver cells were maintained additionally for three or more days on type-1 collagen-coated plates in the continued presence of GM-CSF, they exhibited characteristics of mature DC: MHC class II expression was markedly upregulated, mixed leukocyte reaction stimulatory activity was increased, and phagocytic function was decreased. Similar observations were made when Ia+ cells were depleted from the GM-CSF-propagated cells before exposure to collagen. Further evidence that the GM-CSF-stimulated class IIdim or class II-depleted hepatic NPC were immature DC was obtained by injecting them into allogeneic B10 (H-2b I-E-) recipients. They "homed" to T cell-dependent areas of lymph nodes and spleen where they strongly expressed donor MHC class II antigen 1-5 d later. These observations provide insight into the regulation of DC maturation, and are congruent with the possibility that the migration of immature DC from normal liver and perhaps other organ allografts may help explain their inherent tolerogenicity.

Animals↗
PubMed1994

FK506 inhibits the differentiation of developing thymocytes but not negative selection of T cell receptor V beta 5+ and V beta 11+ T lymphocytes in vivo.

To examine the influence of FK506 on lymphocyte development, we employed a syngeneic bone marrow transplantation model using MHC-disparate B10 (H-2b, I-Ab) and B10.BR (H-2k, I-Ak, I-Ek) mice. B10 mice, which do not express class II I-E, do not delete any known T cell receptor (TCR)-V beta, while B10.BR mice (MHC class II I-Ek, I-Ak) delete V beta 5+ and V beta 11+ TCR. Continuous daily treatment of syngeneically reconstituted B10 mice with FK506 delayed the development of thymocytes from the CD4+CD8+ to CD4+CD8- stage, while no effect was observed at the earlier CD4-CD8- to CD4+CD8+ stage. At the same time, there was a significant reduction in TCRhigh thymocytes compared with untreated, syngeneically reconstituted controls. These results suggest that FK506 treatment interfered with thymic positive selection. We also examined whether FK506 treatment would influence negative selection. Levels of expression of V beta 5+ and V beta 11+ T cells in FK506-treated B10.BR-->B10.BR recipients were similar to those observed in unmanipulated, syngeneically reconstituted B10.BR-->B10.BR controls. This was not due to the inhibition of clonal proliferation by FK506, since 35 days after drug withdrawal complete recovery of the peripheral Thy1.2+ population was observed, while the percentages of V beta 5+ and V beta 11+Thy1.2+ T cells were maintained at values similar to controls. Surprisingly, clonal proliferation stimulated by monoclonal antibody against V beta 5 and V beta 11 TCRs was observed in CsA-treated, syngeneically reconstituted B10.BR mice but not in FK506-treated mice, suggesting that CsA may be more likely to induce autoreactivity. Differences in thymic architecture between FK506- and CsA-treated animals further suggested that the drugs may differ in their effects on T cell development in vivo.

Animals↗
PubMed1994

Chimerism after whole organ transplantation: its relationship to graft rejection and tolerance induction.

Bone marrow-derived "passenger" leukocytes that normally reside in the interstitial tissues of whole organs migrate into the recipient after transplantation and establish microchimerism. An equally important phenomenon is the reciprocal migration of circulating recipient leukocytes, which repopulate the interstitium of whole organ allografts. This bidirectional exchange and interaction of bone marrow-derived cells after organ transplantation is considered a seminal event in the acceptance of allografts and in the induction of donor-specific tolerance. The apparent dichotomous role of donor cell chimerism in the processes of organ rejection and acceptance is discussed.

Bone Marrow Cells↗
PubMed1994

Therapeutic advances in immunosuppression.

Immunosuppressive therapy is appropriate for the prevention or reversal of allograft rejection, and for the treatment of autoimmune disorders and allergic disease. Recent advances in our understanding of the cellular and molecular mechanisms that regulate immune responses have paralleled elucidation of the modes of action of a variety of therapeutic immunosuppressive agents, both 'old' and new. These developments have identified potential targets for more refined and specific intervention strategies that are now being tested in the clinic.

Animals↗
PubMed1994

Circulating intercellular adhesion molecule-1 (ICAM-1) in autoimmune liver disease and evidence for the production of ICAM-1 by cytokine-stimulated human hepatocytes.

A circulating form of the membrane-bound ICAM-1 (CD54), a ligand for lymphocyte function-associated antigen-1 (LFA-1), has recently been identified in normal human serum. In this study, serum levels of soluble ICAM-1 (sICAM-1) were determined by sandwich ELISA both in normal healthy individuals of both sexes and in subjects with autoimmune liver diseases. Patients with primary biliary cirrhosis (PBC), primary sclerosing cholangitis and chronic active hepatitis (autoimmune) showed significant elevations in sICAM-1 compared with normal healthy subjects. The median level in PBC was approximately seven-fold above normal. Significant elevations in sICAM-1 were also detected, however, in patients with inactive alcoholic cirrhosis, suggesting that impaired liver clearance might at least in part account for the increased serum levels seen in patients with autoimmune liver disease. In patients with PBC, sICAM-1 levels were related to summary assessment of disease severity (Child-Pugh classification) and correlated significantly with serum biochemical indices of liver function, including measures both of cholestasis and liver cell injury. In contrast, serum levels of E-selectin did not differ significantly from healthy controls. Although it has been suggested that peripheral blood mononuclear cells (PBMC) may be a source of sICAM-1, investigation of ICAM-1 gene expression by reverse transcriptase polymerase chain reaction revealed similar basal levels of ICAM-1 message in PBMC of normal individuals and those with active PBC. This suggests that PBMC may not be a significant source of sICAM-1 in this disease. Similar increases in ICAM-1 mRNA expression were found in cultured, concanavalin A (Con A)-stimulated lymphocytes of both PBC patients and controls. Significantly, stimulation of cultured, normal human hepatocytes with proinflammatory cytokines and endotoxin induced cell surface expression of ICAM-1 and the secretion/shedding of sICAM-1 into the hepatocyte culture medium. This new finding suggests that hepatocytes may be an important source of sICAM-1 in autoimmune and other chronic liver diseases. The possible role of sICAM-1 in inflammatory disorders remains to be determined.

Adolescent↗
PubMed1994

Circulating proinflammatory cytokines (IL-1 beta, TNF-alpha, and IL-6) and IL-1 receptor antagonist (IL-1Ra) in fulminant hepatic failure and acute hepatitis.

Fulminant hepatic failure (FHF) is characterized by massive necroinflammation of the liver tissue and is associated with high mortality. Serum concentrations of IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-1 receptor antagonist (IL-1Ra) were measured in 30 patients with FHF and in 23 patients with acute hepatitis (AH) before start of treatment and in 23 healthy controls. Levels of all four molecules were increased significantly in FHF compared with AH, in which values were higher than in the healthy controls. High serum levels of IL-1 beta and a significantly reduced ratio of IL-1Ra to IL-1 beta (IL-1Ra/IL-1 beta) were observed in FHF patients who subsequently died compared with subjects who survived. TNF-alpha and IL-6 concentrations were correlated with levels of human hepatocyte growth factor (hHGF), an index of hepatocyte regeneration. Although serum cytokine levels varied considerably between patients within each group studied, it is suggested that the striking elevation in proinflammatory cytokine levels in FHF may reflect both the insufficiency of hepatitis virus elimination and a failure to control a vicious cytokine cascade leading to overwhelming hepatocyte destruction rather than regeneration. The high cytokine levels observed in these patients and the significantly elevated IL-1Ra/IL-1 beta ratio in FHF patients who survived compared with those who did not suggest the possible therapeutic use of cytokine antagonists for the control of this life-threatening disease.

Adolescent↗