Biomedical subjects

A W Thomson

Publications and source records attributed to A W Thomson.

At least 37 records · Page 2Linked to original sources

PubMed2001

Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation.

Although it is known that dendritic cells (DCs) produce cytokines, there is little information about how cytokine synthesis is regulated during DC development. A range of cytokine mRNA/proteins was analyzed in immature (CD86-) or mature (CD86+) murine bone marrow (BM)- derived DCs. Highly purified, flow-sorted, immature DCs exhibited higher amounts of interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor beta1 (TGF-beta1), and macrophage migration inhibitory factor (MIF) mRNA/protein than mature DCs. After differentiation, DC up-regulated the levels of IL-6 and IL-15 mRNA/protein and synthesized de novo mRNA/protein for IL-12p35, IL-12p40, and IL-18. Although immature BM-derived DCs did not stimulate naive allogeneic T cells, mature DCs elicited a mixed population of T helper (Th) 1 (mainly) and Th2 cells in 3d-mixed leukocyte reactions. CD86+ BM DCs switched to different cytokine patterns according to whether they were terminally differentiated by lipopolysaccharide (LPS) or CD40 ligation. Although both stimuli increased IL-6, IL-12p40, IL-15, and TNF-alpha mRNA/protein levels, only LPS up-regulated transcription of IL-1alpha, IL-1beta, IL-12p35, and MIF genes. Although LPS and CD40 cross-linking increased the T-cell allostimulatory function of BM DCs, only LPS stimulation shifted the balance of naive Th differentiation to Th1 cells, a mechanism dependent on the up-regulation of IL-12p35 and not of IL-23. These results demonstrate that, depending on the stimuli used to terminally mature BM DCs, DCs synthesize a different pattern of cytokines and exhibit distinct Th cell-driving potential.

Animals↗

PubMed2001

Interleukin-17 antagonism inhibits acute but not chronic vascular rejection.

BACKGROUND: Blocking the action of interleukin (IL) 17 with an IL-17 receptor (R):Fc fusion protein inhibits T-cell proliferative responses to alloantigens and prolongs vascularized heart graft survival. In this study, we examined whether IL-17 antagonism could suppress the development of chronic rejection. METHODS: A 0.6-cm section of C57BL10 (H2b) thoracic aorta was transplanted to recipient C3H (H2k) abdominal aorta. IL-17R:Fc or control human immunoglobulin G was administered i.p. (500 microg/day) from days 0 to 6 or from days 0 to 29. Mice were killed on days 7 or 30. Grafts were examined histologically and stained for alpha-smooth muscle actin (alpha-smA). Antidonor mixed leukocyte reaction, cytotoxic T cell, and alloantibody responses were quantified. RESULTS: On day 7, control grafts showed mononuclear cell (MNC) infiltration, pronounced endothelial damage, and apoptosis of intimal and medial cell compartments. By day 30, there was concentric intimal thickening, accumulation of alpha-smA+ cells, and collagen deposition. Patchy destruction of the elastic membranes and loss of alpha-smA expression in media were evident. IL-17R:Fc for 6 days decreased MNC infiltration in the intimal and medial compartments at day 7. The endothelium was preserved (completely or partially) in all grafts. The medial compartment showed normal alpha-smA expression. Irrespective of IL-17R:Fc treatment for either 6 days or continuously, allografts harvested at day 30 showed circumferential intimal thickening, with accumulation of alpha-smA+ cells and collagen deposition. There was no effect on circulating alloantibody levels. CONCLUSIONS: These findings support a role for IL-17 in the immunopathogenesis of acute vascular rejection and demonstrate the potential of IL-17 antagonism for therapy. By contrast, IL-17 antagonism does not appear to prevent ensuing chronic graft vascular disease, in particular neointimal formation.

Acute Disease↗

PubMed2001

Aspirin inhibits in vitro maturation and in vivo immunostimulatory function of murine myeloid dendritic cells.

Aspirin is the most commonly used analgesic and antiinflammatory agent. In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC). CD11c and MHC class I expression were unaffected. The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis. Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent. Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-kappaB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-kappaB p50. Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development. The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity. Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis. By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells. They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation. Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue. These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application.

Animals↗

PubMed2001

Mammalian and viral IL-10 enhance C-C chemokine receptor 5 but down-regulate C-C chemokine receptor 7 expression by myeloid dendritic cells: impact on chemotactic responses and in vivo homing ability.

The immunosuppressive and anti-inflammatory cytokine IL-10 inhibits the phenotypic and functional maturation of dendritic cells (DC) and has been reported to confer tolerogenic properties on these important professional APC. Here, we exposed murine bone marrow-derived myeloid DC to either mouse (m) or viral (v) IL-10 early during their in vitro generation in response to GM-CSF and IL-4. Both mIL-10 and vIL-10 down-regulated the expression of CCR7 mRNA determined by RT-PCR, while mIL-10 up-regulated the expression of CCR5 transcripts. These changes in CCR7 and CCR5 expression were associated with inhibition and augmentation, respectively, of DC chemotaxis toward their respective agonists, macrophage inflammatory proteins 3beta and 1alpha, while in vivo homing of DC from peripheral s.c. sites to secondary lymphoid tissue of syngeneic or allogeneic recipients was significantly impaired. Anti-mIL-10R mAb reversed the effects of mIL-10 on CCR expression and restored DC homing ability. Retroviral transduction of mIL-10- and vIL-10-treated DC to overexpress transgenic CCR7 partially restored the cells' lymphoid tissue homing ability in allogeneic recipients. However, CCR7 gene transfer did not reinstate the capacity of IL-10-treated DC to prime host naive T cells for ex vivo proliferative responses or Th1 cytokine (IFN-gamma) production in response to rechallenge with (donor) alloantigen. These findings suggest that in addition to their capacity to subvert DC maturation/function and confer tolerogenic potential on these cells, mIL-10 and vIL-10 regulate DC migratory responses via modulation of CCR expression.

Adjuvants, Immunologic↗

PubMed2001

Differential effects of myeloid dendritic cells retrovirally transduced to express mammalian or viral interleukin-10 on cytotoxic T lymphocyte and natural killer cell functions and resistance to tumor growth.

BACKGROUND: Genetic engineering of dendritic cells (DC) to express immunosuppressive molecule(s) offers potential for therapy of allograft rejection and autoimmune disease. Viral (v) interleukin (IL)-10, encoded by the Epstein-Barr virus, is highly homologous to mammalian (m) IL-10, but lacks certain of its T-cell stimulatory activities. Our aim was to evaluate and compare the influence of vIL-10 and mIL-10 gene transfer on the T-cell and natural killer (NK) cell stimulatory activity of DC, and their impact on the growth of transplantable tumors. METHODS: Myeloid DC progenitors, propagated from the bone marrow of C57BL/6J (H2b) mice in granulocyte-macrophage colony-stimulating factor + IL-4, were transduced using retroviral supematant from the BOSC ecotropic packaging cell line. The function of the IL-b gene-modified DC was assessed by examining their ability to induce naive allogeneic T-cell proliferation and cytotoxic T lymphocyte (CTL) generation. MCA205 (H2b) sarcoma cells mixed with either vIL-10-, mIL-10-, or Zeo (control gene)-transduced DC were inoculated intradermally into C57BL/6J (syngeneic) or BALB/cJ (H2d) (allogeneic) recipients, which were monitored for tumor growth. The role of specific host effector cell populations in tumor resistance was determined by antibody depletion. RESULTS: Compared with control gene-modified DC, both vIL-10- and mIL-10-transduced DC, which secreted the transgene product, showed reduced surface expression of MHC class II and costimulatory molecules, and impaired ability to induce T-cell proliferation. vIL-10-transduced DC were also inhibited with respect to CTL induction but did not affect the generation of NK cells. By contrast, mIL-10-transduced DC augmented CTL generation and NK cell activity. In the tumor transplant model, vIL-10-transduced DC enhanced tumor growth both in syngeneic and allogeneic hosts, whereas mIL-10-transduced cells inhibited tumor development. Depletion of CD4+ or CD8+ T cells or NK cells in mice given mIL-10-transduced DC reversed this therapeutic effect. CONCLUSION: mIL-10 gene-modified myeloid DC promote CTL and NK cell-mediated responses and inhibit tumor growth. By contrast, vIL-10-engineered DC, which elicit diminished CTL responses and do not promote NK cell activity, seem to have therapeutic potential for inhibition of T cell-mediated immunity.

Animals↗

PubMed2001

Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway.

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.

Adenosine Triphosphate↗

PubMed2001

Il-12 antagonism enhances apoptotic death of T cells within hepatic allografts from Flt3 ligand-treated donors and promotes graft acceptance.

Mouse livers are accepted across MHC barriers and induce donor-specific tolerance without immunosuppressive therapy. By contrast, livers from donors treated with Flt3 ligand, which dramatically increases hepatic interstitial dendritic cells, are rejected acutely (median survival time 5 days). This switch from tolerance to rejection is associated with a marked reduction in apoptotic activity of graft-infiltrating cells. We hypothesized that IL-12 production by enhanced numbers of donor APC might inhibit apoptosis, promote expansion of Th1 cells, and play a key role in liver rejection. Therefore, C3H (H2(k)) recipients of liver grafts from Flt3 ligand-treated B10 donors were given neutralizing anti-IL-12 mAb (200 or 500 microg) on days 0 and 2 after transplant. Graft survival was markedly prolonged at the higher mAb dose, with 50% of grafts surviving >100 days. This effect was associated with reductions in IFN-gamma gene transcripts within the graft-infiltrating cell population and with reductions in circulating IFN-gamma and IL-10 levels, donor-specific CTL and NK cell activities, and circulating alloantibody levels. At the same time, there were marked increases in apoptotic (TUNEL(+)) CD4(+) and especially CD8(+) cells, both within the grafts and in spleens of anti-IL-12 mAb-treated mice. In vitro, exogenous IL-12 inhibited apoptotic death induced in naive allogeneic T cells by liver nonparenchymal cells. These findings suggest that suppression of rejection by IL-12 antagonism, linked to restoration of apoptotic activity within the peripheral alloreactive T cell population, is important for liver allograft survival and tolerance induction.

Adjuvants, Immunologic↗

PubMed2001

Flt3 ligand pretreatment promotes protective immunity to Listeria monocytogenes.

Flt3 ligand (Flt3L) plays a critical role in the proliferation, differentiation and survival of haematopoietic progenitor cells. Its potential use in a clinical setting has been suggested. Here, we report that mice administered Flt3L displayed a nine-fold increase in size of their hepatic non-parenchymal cell population and an approximate 365-fold increase in number of mature dendritic cells within their livers. Such mice exhibited an elevated resistance to secondary infections with Listeria monocytogenes, an intracellular bacterial pathogen. More than 2.0 log(10)fewer listeriae were recovered in the livers of Flt3L-treated, than untreated, mice on day 2 following secondary challenge. Importantly, Flt3L-pretreated mice immunized with an avirulent (listeriolysin O-negative) strain of Listeria harbored significantly fewer ( approximately 1.5 log(10)) organisms in their spleens and livers than did control mice immunized with listeriolysin O-negative listeriae and challenged with a lethal dose of bacteria. The latter finding supports a potential role for Flt3L in strategies to develop vaccines to intracellular pathogens.

Adjuvants, Immunologic↗

PubMed2001

Viral IL-10 gene transfer inhibits DTH responses to soluble antigens: evidence for involvement of genetically modified dendritic cells and macrophages.

Expression of the viral interleukin-10 (vIL-10) gene within one joint of an animal with polyarticular, inflammatory arthritis suppresses disease in both treated and untreated joints (the "contralateral effect"). We used a mouse delayed-type hypersensitivity (DTH) model to investigate this phenomenon. Adenoviral delivery of the vIL-10 gene suppressed DTH reactions in injected and contralateral paws. T lymphocytes recovered from immunized mice injected with the adenoviral vector (ad-vIL-10) were unable to transfer the DTH response, but were not inhibitory. Peritoneal exudate cells recovered from mice injected intraperitoneally with ad-vIL-10 inhibited DTH reactions in recipient mice, but only when the donor mice had been sensitized to the antigen used to incite the DTH response. Dendritic cells (DCs) recovered from the draining lymph nodes of mice injected with ad-vIL-10 behaved similarly. Bone-marrow-derived DCs cultured ex vivo with ad-vIL-10 or recombinant mouse IL-10 also suppressed DTH reactions by adoptive transfer when pulsed with the inciting antigen. Collectively, these data suggest a mechanism for the contralateral effect in which genetically modified macrophages and DCs present antigen in the context of high, local concentrations of vIL-10, thereby generating unresponsive T lymphocytes. These findings suggest new ways in which to treat immune-driven diseases by gene and cell therapy.

Adenoviridae↗

PubMed2001

Potential of tolerogenic dendritic cells for transplantation.

Dendritic cells (DC) are professional antigen (Ag)-presenting cells considered traditionally as the passenger leukocytes that, after migration from transplanted tissues, stimulate allospecific naive T cell responses and trigger acute rejection. However, there is recent evidence that, besides their role in central T lymphocyte deletion in the thymus, DC perform a crucial function to induce/maintain peripheral T cell tolerance. This paper outlines conceptual models that try to explain how DC may induce/maintain tolerance. It also considers how such ideas have been implemented recently in an effort to generate tolerogenic DC to induce donor Ag-specific tolerance/ immunosuppression and prolonged allograft survival. These approaches include genetic engineering of donor- or recipient-derived DC to express molecules capable of promoting tolerance to alloAg.

Animals↗

PubMed2001

Designer dendritic cells for tolerance induction: guided not misguided missiles.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play crucial roles as initiators and modulators of adaptive immune responses. Although DC-based vaccines have been utilized successfully to generate cytolytic T-cell activity against tumor antigens (Ags), evidence has accumulated that DCs also have potent capabilities to tolerize T cells in an Ag-specific manner. DCs cultured in the laboratory can suppress auto- or alloimmunity. Current and prospective strategies to promote this inherent tolerogenic potential of DCs might prove to be important for the therapy of transplant rejection and autoimmune diseases.

Animals↗

PubMed2001

Dermal-resident CD14+ cells differentiate into Langerhans cells.

Epidermal Langerhans cells (LCs) show extraordinary immunostimulatory capacity and play a key role in the initiation and regulation of immune responses. Studies of LC biology are currently the focus of efforts to engineer immune responses and to better understand the immunopathology of cutaneous diseases. Here we identified and characterized a population of LC precursors that were resident in human skin. These immediate precursors expressed CD14, langerin and functional CCR6. When cultured with transforming growth factor-beta1 alone, they had the potential to differentiate into epidermal LCs; when cultured in the presence of granulocyte macrophage-colony-stimulating factor and interleukin 4 they differentiated into functionally mature dendritic cells. Identification and characterization of these LC precursors provided insight into LC biology and the mechanism(s) through which LCs repopulate the epidermis.

Antigens↗

PubMed2000

Inhibition of the allograft response by donor specific blood transfusion: association with reduced local TH1 cytokines and nitric oxide but enhanced prostaglandin E2 production.

BACKGROUND: Donor-specific blood transfusion (DST) may improve allograft survival in human and animal models, but the mechanisms for this graft protective effect are incompletely understood. The sponge matrix allograft model was used to determine if DST induces regulatory factors within the allograft. METHODS: C57BL/6 (H-2b) recipients received donor-specific (DBA/2J, H-2d) or syngeneic (C57BL/6) blood 7 days before sponge matrix allograft (DBA/2J) implantation. Fourteen days postgrafting, the sponge infiltrating cells (SIC) were examined for cytotoxic T cell (CTL) and natural killer (NK) activity, and sponge exudate fluid (SEF) was assessed for nitric oxide (.N=O) and prostaglandin E2 (PGE2) content. Interleukin- (IL) 2, IL-4, IL-10, and interferon-gamma (IFN-gamma) production by SIC was also determined. Recipient splenocytes were simultaneously assessed for anti-donor cytotoxic and proliferative responses and .N=O production. RESULTS: SIC from mice receiving syngeneic transfusions (ST) acquired both CTL and NK activity postgrafting, with maximal activity by day 14. DST suppressed both CTL and NK activity throughout the postgrafting period. Limiting dilution analysis (LDA) of SIC to determine precursor and native CTL frequency showed significantly lower responder cell frequency after DST compared with ST. SEF .N=O levels and SIC production of IL-2 and IFN-gamma in grafted DST mice were significantly lower than in grafted mice receiving ST. No significant amounts of IL-4 and very low levels of IL-10 were produced by SIC from grafted mice after either ST or DST. Conversely, PGE2 content of sponge fluid and serum from DST mice was higher than in mice receiving ST. Antigen stimulated splenocyte proliferation and CTL development assessed by LDA were also inhibited by DST. CONCLUSIONS: Reduction in local TH1 cytokines, absence of detectable TH2 cytokines, with enhanced PGE2 and depressed .N=O were observed in the local graft environment after DST. These data support the hypothesis that DST induces donor-specific intragraft suppressor factors, accompanied by reduced local and systemic immune activation.

Animals↗

PubMed2000

Phenotypic and functional characterization of mouse hepatic CD8 alpha+ lymphoid-related dendritic cells.

Recently, attention has focussed on phenotypic and functional differences between classic myeloid dendritic cells (DC), and DC that reportedly develop from an early, committed lymphoid precursor. In mice, DC from these separate hemopoietic lineages differ by their surface expression of CD8 alpha. We undertook a comparative study of CD8 alpha+ (CD11blow; lymphoid-related) and CD8 alpha- (CD11bhigh; myeloid) DC isolated from mouse liver. CD8 alpha+ and CD8 alpha- DC each constituted </=1.0% of the freshly isolated, normal nonparenchymal cells (NPC). Both populations were enriched 10-15% by overnight culture and metrizamide density centrifugation. Flt3 ligand (Flt3L) potently induced equal expansion of both subsets in vivo. Tissue-resident CD8 alpha+ DC, freshly isolated from Flt3L-treated mice, existed primarily as immature cells (CD11c+, CD11blow, CD40-/low, CD80low, CD86low, MHC class IIlow), consistent with previous observations regarding bulk DC freshly isolated from nonlymphoid tissues. Following overnight culture in GM-CSF, CD8 alpha+ DC underwent phenotypic and functional maturation equivalent to that observed for CD8 alpha- DC. CD95 ligand (FasL) mRNA was detected in both immature and mature DC of each subset. In vitro analysis confirmed that flow-sorted, mature CD8 alpha+ and CD8 alpha- DC were strong and equally efficient stimulators of allogeneic T cell proliferation in primary MLR. Both immunohistochemical and genomic DNA analysis revealed that in vivo, sorted CD8 alpha+ DC trafficked from s.c. sites to T cell areas of allogeneic lymphoid tissue and were equally efficient at priming naive T cells compared with CD8 alpha- DC. This is the first comparative study of lymphoid-related DC isolated from nonlymphoid tissue.

Adoptive Transfer↗

PubMed2000

Microchimerism, donor dendritic cells, and alloimmune reactivity in recipients of Flt3 ligand-mobilized hemopoietic cells: modulation by tacrolimus.

Flt3 ligand (FL) is a potent hemopoietic growth factor that strikingly enhances stem cells and dendritic cells (DC) in vivo. We examined the impact of infusing FL-mobilized bone marrow (BM) cells on microchimerism and anti-donor reactivity in normal and tacrolimus-immunosuppressed, noncytoablated allogeneic recipients. BM from B10 (H2b) mice given FL (10 microg/day; days 0-8; FL-BM) contained a 7-fold higher incidence of potentially tolerogenic immature CD11c+ DC (CD40low, CD80low, CD86low, MHC IIlow) that induced alloantigen-specific T cell hyporesponsiveness in vitro. C3H (H2k) mice received 50 x 106 normal or FL-BM cells (day 0) and tacrolimus (2 mg/kg/day; days 0-12). On day 15, enhanced numbers of donor (IAb+) cells were detected in the thymi and spleens of FL-BM recipients. Tacrolimus markedly enhanced microchimerism, which declined as a function of time. Ex vivo splenocyte proliferative and CTL responses and Th1 cytokine (IFN-gamma) production in response to donor alloantigens were augmented by FL-BM infusion, but reduced by tacrolimus. Systemic infusion of purified FL-BM immature DC, equivalent in number to that in corresponding whole BM, confirmed their capacity to sensitize, rather than tolerize, recipient T cells in vivo. In vitro, tacrolimus suppressed GM-CSF-stimulated growth of myeloid DC from normal BM much more effectively than from FL-BM without affecting MHC class II or costimulatory molecule expression. Infusion of normal B10 BM cells at the time of transplant prolonged C3H heart allograft survival, whereas FL-BM cells did not. A therapeutic effect of tacrolimus on graft survival was observed in combination with normal, but not FL-BM cells. These findings suggest the need for alternative immunosuppressive strategies to calcineurin inhibition to enable the engraftment, survival, and immunomodulatory function of FL-enhanced, immature donor DC.

Adjuvants, Immunologic↗

PubMed2000

Preferential induction of Th1 responses by functionally mature hepatic (CD8alpha- and CD8alpha+) dendritic cells: association with conversion from liver transplant tolerance to acute rejection.

BACKGROUND: Liver grafts are accepted across major histocompatibility barriers in mice without immunosuppressive therapy. Potentially tolerogenic immature donor dendritic cells (DC) may play a key role in this phenomenon, but recovery of purified DC from normal livers for functional analysis is inherently difficult. Administration of in vitro propagated immature donor DC to recipients of different types of allograft can prolong transplant survival. By contrast, marked increases in donor liver DC as the result of Flt3 ligand (FL) administration and the resulting augmentation of allostimulatory activity within host lymphoid tissue, is associated with acute graft rejection. Here, we compared the capacity of in vitro generated normal liver immature DC and FL-treated donor liver DC to induce alloimmune CD4+ T helper (Th) 1/Th2 and CD8+ T cytotoxic (Tc) 1/Tc2 responses, in vitro and in vivo. METHODS: B10 (H2b, IAb) immature liver DC were propagated from normal hepatic nonparenchymal cells in granulocyte macrophage-colony stimulating factor (GM-CSF) for 6-8 days. Freshly isolated DC from livers of FL-treated mice (FL-liver DC) were cultured overnight (o/n) in GM-CSF, and both myeloid (CD11c+ CD8alpha-) and lymphoid DC (CD11c+ CD8alpha+) flow-sorted for functional analysis. Proliferative activity and production of interferon (IFN)-gamma, interleukin (IL)-4, and IL-10 by naive C3H (H2k, IEk) T cells in response to DC stimulation was assessed by [3H]thymidine incorporation, and by multicolor flow cytometric analysis, respectively, after 3-day mixed leukocyte reactions. To investigate their in vivo trafficking, B10 DC were injected subcutaneously into normal C3H mice. Sections of lymphoid tissue were immunostained for donor MHC class II+ (IAb+) cells, and for IFN-gamma, IL-4, and IL-10 production. Donor cells and clusters of specific cytokine-secreting cells were enumerated. RESULTS: Both in vitro propagated normal liver-derived DC, and freshly isolated bulk FL-liver DC showed an immature phenotype (MHC class II(lo), CD40-, CD80-, and CD86-) and were weak stimulators of naive allogeneic T cells. After o/n incubation in GM-CSF, both CD8alpha- and CD8alpha+ FL-liver DC exhibited marked up-regulation of surface MHC class II and costimulatory molecules, and acquired potent stimulatory activity for Th1 (mainly) and Th2 cells. Both in vitro propagated immature DC and o/n-cultured mature FL-liver DC homed in vivo to host lymphoid tissues, but with different kinetics. Whereas the mature allogeneic FL-liver DC induced IFN-gamma+ clusters in splenic T-cell areas within 2 days, the IFN-gamma response to immature DC was much slower and weaker. CONCLUSIONS: FL-treated donor livers that are rejected acutely contain markedly enhanced numbers of myeloid (CD8alpha-) and lymphoid (CD8alpha+) DC, many of which are capable of maturing rapidly into strong inducers of Th1 and Tcl responses. Substantial differences in quantity, and both the phenotypic and functional characteristics of the DC constituency of donor livers, may contribute significantly toward the distinct outcomes of liver transplant tolerance and rejection.

Acute Disease↗

PubMed2000

Effects of liver-derived dendritic cell progenitors on Th1- and Th2-like cytokine responses in vitro and in vivo.

There is evidence that donor-derived dendritic cells (DC), particularly those at a precursor/immature stage, may play a role in the immune privilege of liver allografts. Underlying mechanisms are poorly understood. We have examined the influence of in vitro generated mouse liver-derived DC progenitors (DCp) on proliferative, cytotoxic, and Th1/Th2 cytokine responses induced in allogeneic T cells. Liver DCp, propagated in GM-CSF from C57B10 mice (H2b), induced only minimal proliferation, and weak cytotoxic responses in allogeneic (C3H; H2k) T cells compared with mature bone marrow (BM)-derived DC. Flow-cytometric analysis of intracellular cytokine staining revealed that mature BM DC, but not liver DCp, elicited CD4+ T cell production of IFN-gamma. Intracellular expression of IL-10 was very low in both BM DC- and liver DCp-stimulated CD4+ T cells. Only stimulation by liver DCp was associated with IL-10 secretion in primary MLR. Notably, these liver DCp cocultured with allogeneic T cells stained strongly for IL-10. Following local (s.c. ) injection in allogeneic recipients, both BM DC and liver DCp homed to T cell areas of draining lymph nodes and spleen, where they were readily detected by immunohistochemistry up to 2 wk postinjection. Liver DCp induced clusters of IL-10- and IL-4-secreting mononuclear cells, whereas Th2 cytokine-secreting cells were not detected in mice injected with mature BM DC. By contrast, comparatively high numbers of IFN-gamma+ cells were induced by BM DC. Modulation of Th2 cytokine production by donor-derived DCp may contribute to the comparative immune privilege of hepatic allografts.

Animals↗

PubMed2000

Flt3 ligand (FL) and its influence on immune reactivity.

Flt3 (fms-like tyrosine kinase 3) ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of progenitor and stem cells both in vivo and in vitro. Its capacity to augment strikingly the numbers of dendritic cells (rare antigen-presenting cells that induce and regulate immune responses) in mice and humans has stimulated considerable interest in its value as an investigational tool and therapeutic agent. In this review, we survey the hematopoietic properties and immunobiology of FL, and examine its therapeutic potential.

Animals↗