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Biomedical subjects

S C Knight

Publications and source records attributed to S C Knight.

At least 19 recordsLinked to original sources
PubMed1999

Interleukin-12 administration in retroviral infection of mice increases the potential to produce functional dendritic cells from bone marrow stem cells.

Rauscher leukaemia virus (RLV) infection in mice causes production of lymph node and skin dendritic cells (DC) that fail to stimulate a primary mixed leukocyte reaction (MLR). Treatment of mice with IL-12 around the time of infection results in DC with normal stimulatory function (N.J. Williams, J.J. Harvey, I. Duncan, R.F.G. Booth, S.C. Knight, Cell Immunol. 183 (1988) 121-130). Here we derived DC from mouse bone marrow by culture with granulocyte macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha) for 10-12 days; DC were generated from bone marrow cells taken from normal mice, from mice injected 15 days earlier with RLV or from those receiving RLV plus five daily doses of 100 ng of IL-12 starting 2 days before infection. Infection of the DC with RLV was assessed from nested PCR with doubling dilutions of DNA and the capacity of DC to stimulate a MLR was tested. DC derived from bone marrow of IL-12 treated animals showed at least twice the level of infection with RLV as those from non-treated animals although infection never exceeded 20% of the cells. DC derived from bone marrow of mice given RLV caused negligible stimulation of the MLR but those from mice additionally treated with IL-12 functioned normally. Thus, treatment of mice with IL-12 promoted the potential of stem cells taken 12 days after the last IL-12 injection to develop into functional DC despite increased infection with virus. Treatment of mice with IL-12 may have a long term effect on the potential growth of DC from stem cells which may contribute to the potency of this cytokine in promoting cell mediated immune responses.

Animals↗
PubMed1998

Interleukin-12 restores dendritic cell function and cell-mediated immunity in retrovirus-infected mice.

The effects of IL-12 treatment on the defects in DC function and on the reduced cell-mediated immunity induced in mice infected with Rauscher leukemia virus (RLV) were studied. DC from RLV-infected mice failed to stimulate significant allogeneic T cell proliferation but T cells from RLV-infected mice showed normal responses to allogeneic DC. In RLV-infected mice treatment with 5 doses of 100 or 300 ng IL-12 around the time of infection resulted in DC that stimulated normal T cell proliferation. Treatment of mice with 300 ng IL-12 but not 100 ng reduced T cell responses. RLV-infected mice showed reduced delayed hypersensitivity to a contact sensitizer. Infected animals receiving the low dose of IL-12 which allowed normal DC and T cell function gave normal delayed hypersensitivity reactions; IL-12 thus resulted in both normal T cell stimulation by DC and cell-mediated immunity. A failure of T cell stimulation by DC is associated with immunosuppression in retrovirus infection and the enhanced capacity of DC to stimulate T cells after IL-12 treatment may be beneficial.

Animals↗
PubMed1998

Transfer of antigen between dendritic cells in the stimulation of primary T cell proliferation.

Primary proliferative T cell responses require stimulation with antigen-pulsed dendritic cells (Ag-DC). Here we show that for optimal stimulation, dendritic cells (DC) not exposed directly to antigen are also required. Ag-DC added to DC-depleted T cells caused negligible primary stimulation; adding back DC resulted in stimulation. These effects were seen using the contact sensitizer fluorescein isothiocyanate (FITC), FITC conjugated to ovalbumin (FITC-OVA) or influenza virus as antigens. DC co-cultured with Ag-DC (using FITC or FITC-OVA) acquired antigen indicating that antigen was transferred between DC. DC that acquired antigen secondarily were separated by cell sorting and stimulated primary T cell proliferation directly. DC were also pulsed with FITC, washed thoroughly and incubated overnight. Supernatants contained shed antigen since DC incubated in these supernatants acquired antigen as indicated by flow cytometry. DC acquiring the shed antigen also stimulated T cell proliferation although the stimulation was not as effective as that seen when cell contact between DC and antigen-bearing DC occurred. Thus, in primary stimulation, activation of T cells may occur when there is an antigen gradient between Ag-DC and DC and the mechanisms underlying these effects are now being sought. We propose that this unique interaction between antigen-presenting cells may be a paradigm for self/non-self discrimination.

Animals↗
PubMed1998

Increase in dendritic cell numbers, their function and the proportion uninfected during AZT therapy.

The effects of AZT treatment on the numbers, level of infection and function of peripheral blood dendritic cells (DC) were examined in patients with HIV infection. This was a cross-sectional study of patients before AZT treatment and up to 20 months after initiation of treatment. Numbers of DC separated by density gradients were below the normal range in patients before treatment, but increased between 3 and 12 months of treatment. The numbers of DC per provirus copy rose from around 100 cells to 5000 cells and this decrease in viral load in DC was significant between 3 and 20 months of treatment. The capacity of DC to stimulate allogeneic T cell proliferation was low before treatment and significantly higher between 6 and 12 months after the start of AZT. This study indicated that AZT treatment produced beneficial effects on DC by increasing their numbers, reducing the provirus load and increasing their function in stimulating T cells. These results support the thesis that the function of these potent antigen-presenting cells is important in development of immunological defects in AIDS, and that effects of AZT treatment on DC may provide a measure of its therapeutic effect.

Anti-HIV Agents↗
PubMed1998

IL-12 increases CD80 expression and the stimulatory capacity of bone marrow-derived dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells derived from CD34 bone marrow stem cells. They undergo a series of maturational steps that allow them to stimulate primary T cell responses. Several cytokines are known to contribute to this process. In this study murine DC maturing from bone marrow progenitors under the influence of granulocyte macrophage colony stimulating factor and tumour necrosis factor-alpha were found to produce IL-12 as measured by ELISA and by flow cytometry to detect intracellular cytokine. Administration of additional IL-12 from day 3 to 7 of culture altered the function and phenotype of DC; enhanced stimulation of T cell proliferation by DC in allogeneic mixed leukocyte reactions was associated with an increase in the surface expression of CD80 on DC. These effects were dose dependent, and were consistently seen with IL-12 at 25 ng/ml and were less marked with IL-12 at 50 ng/ml. These results show that IL-12 is both produced by DC and can increase their stimulatory capacity. The findings suggest that there may be an autocrine effect of IL-12 on DC maturation and function.

Age Factors↗
PubMed1998

Analysis of human immunodeficiency virus type 1 (HIV-1) variants and levels of infection in dendritic and T cells from symptomatic HIV-1-infected patients.

Dendritic cells (DC) are required to initiate primary cellular immune responses. Human immunodeficiency virus type 1 (HIV-1) infection of DC may be central to transmission and persistence of virus and in the pathogenesis of AIDS. In symptomatic HIV-1-infected patients the proportion of DC in the mononuclear cell population was reduced. Provirus load in the T cells was 3-100 times higher than in DC and there was no correlation between the levels of infection in the two cell types. Phylogenetic analysis of amino acids in the V3 loop and flanking regions indicated intermingling of sequences and thus provides the first evidence for transfer of virus between DC and T cells in vivo. In one of three patients analysed there were significant differences in amino acid residues in the V3 region. This may reflect reduced interactions between DC and T cells in infected individuals and for the existence of variants with a stronger tropism for DC, which could play a role in transmission by initiating infection in mucosal DC.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Protection against ascending infection of the genital tract by Chlamydia trachomatis is associated with recruitment of major histocompatibility complex class II antigen-presenting cells into uterine tissue.

A mouse model of ascending infection following intravaginal inoculation with a strain of Chlamydia trachomatis isolated from humans has been used to identify immune mechanisms associated with protection against genital infection. BALB/c and C3H mice differed in their susceptibilities to infection and inflammatory disease. In both mouse strains, ascension of the organism and recruitment of bone marrow-derived mononuclear leukocytes were evident in uterine tissue 1 week postinfection. By 3 weeks the organism had been cleared and inflammation had been resolved in the BALB/c mice, but both persisted in the C3H animals. In athymic nude BALB/c mice both the organism and inflammation persisted, indicating the influence of the hosts' immune response on the outcome of infection. Both BALB/c and C3H mice had a Th1 response in draining lymph nodes, with predominant production of gamma interferon and tumor necrosis factor alpha, low levels of interleukin-10, and no detectable levels of interleukin-4. However, the composition of the early uterine infiltrate differed in these two mouse strains. Cell surface labeling and analysis of light scatter properties by flow cytometry identified a population of large, CD45(+) major histocompatibility complex class II mononuclear cells, which were a prominent feature of the infiltrates in BALB/c mice but were present in significantly lower numbers in C3H mice. These cells expressed the costimulatory molecules CD86 and CD40 and stimulated allogeneic T cells, suggesting that these mononuclear cells are a population of antigen-presenting cells and that they may play a role in clearing antigen and protecting against inflammatory disease in BALB/c mice. An additional level of immunological control may thus exist in genital chlamydial infection.

Animals↗
PubMed1998

The in-vitro generation of dendritic cells from blast cells in acute leukaemia.

Dendritic cells (DC) are potent antigen-presenting cells responsible for the initiation of primary antigen-specific immune responses. In chronic myeloid leukaemia DC have been generated from Ph+ cells and these Ph+ DC are capable of stimulating cytolytic T-cell responses against the parent leukaemia cells. The prevalence of this phenomenon in acute leukaemia (AL) is unknown and we have therefore studied a variety of acute leukaemias to determine their potential for DC development. Peripheral blood mononuclear cells (PBMC) from 21 cases of AL were cultured in GM-CSF + TNF alpha. Of these cases, 15 were viable in culture and cells with typical DC morphology were observed in 12 of these 15 cases. DC growing in culture expressed either CDla and/or CD83 and were HLA-DR+ CD40+ CD80+ CD86+ typical of mature DC. In 9/12 cases the cultured cells possessed potent antigen-presenting capacity as measured in the allo-MLR. The malignant origin of the cultured DC was confirmed by FISH analysis in two cases (one 5q- and one Ph+ AL) and by persistent aberrant expression of CD19 in two cases of biphenotypic leukaemia. Functional DC may be derived from AL blasts in a significant number of patients and such DC may be capable of inducing leukaemia-specific immune responses with potential for clinically beneficial effects.

Acute Disease↗
PubMed1997

Primary proliferative responses to peptides of HIV Gag p24.

Primary proliferative responses can be initiated by adding dendritic cells pulsed with antigen to autologous T cells in 20-microliter hanging drop cultures. To identify primary T-cell epitopes of HIV gag, a series of 23 overlapping peptides, 15 amino acids long, spanning the p24 region were used. Significant proliferative responses were induced in cells from healthy HIV-negative donors by 11 of these peptides. One of two peptides that bound human leukocyte antigen (HLA)-A *0201 in a peptide-binding assay using the antigen-processing defective cell line T2 also induced a primary proliferative response. Primary T-cell proliferation was seen in response to some peptides of gag that have not previously been identified as T-cell epitopes in cells from infected individuals. These epitopes might be useful not only for vaccines in antigenically naive individuals but also might increase the breadth of immune responses in seropositive patients.

Amino Acid Sequence↗
PubMed1997

Mechanisms of loss of functional dendritic cells in HIV-1 infection.

Dendritic cells (DC) are lost from blood and skin during injection with HIV-1; those remaining show a reduced capacity to stimulate T cell proliferation [S. C. Knight, AIDS 10, 807-817]. Our recent studies investigate mechanisms underlying these effects. DC exposed to HIV-1 vitro can act as targets for cytotoxic T cells, although optimal killing was not obtained until DC were exposed to HIV-1 for 3 days. This cytotoxicity may provide a feedback mechanism by which DC that have presented antigens are removed. However, this effect could also contribute to the reduction in DC during persistent infection. We have also investigated the effect of exposure to HIV-1 on DC function. DC exposed to HIV-1 IIIB virus for 2 h stimulated primary proliferative and cytotoxic T cell responses in vitro; these effects may be similar to those occurring during the early activation of protective antiviral immunity in vivo. After exposure of DC to virus for 5 days, stimulation of allogeneic T cells was reduced. However, a different situation applied when using DC developed from CD34+ cord blood stem cells under the influence of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha that were exposed at 24 h to the same virus. These DC showed low levels of infection similar to peripheral blood DC but in contrast stimulated normal allogeneic T cell proliferation. The capacity of DC exposed to HIV-1 to stimulate T cell proliferation or to show a blocked stimulatory capacity may thus depend not only on the length of the exposure to virus but also on the maturational state of the DC. Loss in DC numbers and function on exposure to HIV-1 may result in lower levels of stimulation of T cells, which in turn may be instrumental in reduction of T cell numbers.

Cell Differentiation↗
PubMed1997

Dendritic cells as targets for cytotoxic T lymphocytes.

Dendritic cells (DC) carry antigen into lymph nodes where they may cluster with CD4 and CD8+ lymphocytes and activate both subsets in the initiation of immune responses. Since DC do not leave the lymph nodes in the efferent lymph they may die within the lymph nodes. Another possibility is that they are targets for cytotoxic T cells (CTL) when expressing appropriate epitopes. This possibility was tested in vitro using human peripheral blood DC to stimulate the development of primary CTL in response to HIV-1 or one of its T-cell epitopes (e.g. env 111-126) and secondary CTL in response to type A influenza virus. Pooled CTL generated during six day cultures in 60 replicate 20 microliters hanging drops were tested in a conventional CTL assay. The HIV or HIV peptide stimulated CTL lysed HIV infected DC while the influenza-virus induced CTL killed DC targets infected with this virus. DC were not lysed significantly until they had been exposed to virus for 2-3 days and thus are not highly susceptible to lysis. However, killing of DC after 2-3 days infection with virus may be a feedback mechanism for removing antigen presenting cells after they have stimulated T cell responses. Removal of persistently infected DC by CD8+ CTL may also contribute to the reduction in DC numbers observed in blood and skin in HIV infection.

Antigens, Viral↗
PubMed1997

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules.

To investigate the binding properties of dendritic cells (DC) to vascular endothelium, a comparative analysis was undertaken of DC, monocytes and lymphocytes isolated from the blood of 25 healthy subjects using monolayers of human umbilical vein endothelial cells as the adherence substrate. More blood DC (mean 24% adherence) were adherent to endothelial monolayers than monocytes (mean 18%; P < 0.001) and lymphocytes (mean 12%; P < 0.001). When the monolayers were pretreated with tumour necrosis factor-alpha (TNF-alpha) all leucocyte populations exhibited an increased attachment, but there was still greater binding of DC (mean 37% adherence) in comparison with monocytes (mean 23%; P < 0.001) and lymphocytes (mean 18%; P < 0.001). Flow cytometric analysis revealed that in relation to monocytes and lymphocytes the DC had a higher surface expression of the adhesion molecules CD11a (P < 0.05), CD11c (P < 0.005) and CD54 (P < 0.005) but a lower prevalence of cells bearing CD49d (mean 38%; P < 0.05) and the homing receptor CD62L (mean 14%; P < 0.001). CD1a was present on 22% of DC and virtually absent from the surface of monocytes and lymphocytes. The intensity of expression of the beta1-integrins, CD49c, CD49d and CD49e was greater on DC than lymphocytes and monocytes (P < 0.05). Antibody blocking studies demonstrated that DC binding to untreated and TNF-alpha-treated endothelium was dependent upon the expression of CD11a, CD18 and CD49d, and the simultaneous application of anti-CD18 and anti-CD49d antibodies produced an approximate 70% inhibition of adhesion (P < 0.001). Thus, the expression of both beta1- and beta2-integrins contributes to the adhesive interaction between DC and endothelium.

Cell Adhesion↗
PubMed1997

Adhesion molecules are upregulated on dendritic cells isolated from human blood.

This study investigated whether the high expression of adhesion molecules on enriched preparations of circulating dendritic cells (DCs) was an intrinsic property of the cells or whether it was a consequence of the procedure used to isolate them from blood. Expression of the beta 1, beta 2 integrins (CD11/CD18 family) and other adhesion molecules on DCs in whole blood was compared with that on isolated DCs. Dendritic cells were identified by flow cytometry as leucocytes that were positive for human leucocyte antigen (HLA)-DR, but negative for CD3, CD14, CD16, CD19 and CD56. In contrast to a minority of DCs in whole blood, the majority of isolated DCs expressed the beta 2 integrins and there were a greater number of cells bearing CD44, CD54 and some of the beta 1 integrins (notably CD49b, CD49d, CD49e and CD29). An increase in the proportion of DCs bearing adhesion molecules was generally apparent at the isolation stage when mononuclear cells, which had been incubated overnight, were centrifuged on a metrizamide gradient to enrich for cells of low density. Inclusion of an inhibitor of protein glycosylation and exocytosis (brefeldin A) at all stages of separation partially prevented an increase in the percentage of DCs bearing CD18, C29 and C54 whereas the inclusion of cycloheximide (an inhibitor of polypeptide synthesis) interfered with increases in the percentage of cells bearing CD29 and CD54. Neither of these antagonists had an effect on the intensity of adhesion molecule expression. We suggest that some of the adhesion-dependent functions of isolated DCs are caused, in part, by an upregulation of surface adhesion molecules induced by the enrichment procedure.

Brefeldin A↗
PubMed1997

Bone marrow-derived dendritic cells, infection with human immunodeficiency virus, and immunopathology.

Dendritic cells (DC) exposed to HIV-1 show nonproductive infection that may become productive as they mature. The distribution of DC within genital mucosa and their susceptibility to infection particularly with clade E viruses could be reflected in the ease of heterosexual transmission. Carriage of virus and viral antigen by DC into lymph nodes may allow clustering and activation of T cells and production of protective immune responses. However, secondary infection of activated T cells from infected DC could cause dissemination of virus and loss of infected DC and T cells. In asymptomatic infection, fewer dendritic cells with reduced capacity to stimulate CD4 T cell proliferation are found before evidence of T cell abnormalities, and these early changes in antigen-presenting cells may result in a decline in the production of CD4 memory T cells. However, DC fuel ongoing production of antibody to HIV-1. Signaling by DC to T cells may thus underlie two major features of early HIV infection--loss in CD4+ memory T cells and persistence of antibody production. In AIDS, infected dendritic and epithelial cells within the thymus may affect maturation and contribute to loss of the "naive" T cell population. Further loss of memory T cells may occur through syncytium formation with infected DC. Finally, in AIDS patients, there is a failure in the development and the function of DC from CD34+ stem cells. In conclusion, the infection of dendritic cells, loss in their numbers, and changed signaling to T cells may shape the pattern of immunity during infection with HIV-1. Conversely, treatments that reverse the defect in antigen presentation by DC may improve cell-mediated immunity.

Animals↗
PubMed1996

Mechanism for dendritic cell dysfunction in retroviral infection of mice.

Dendritic cells (DC) from mice infected with the murine retrovirus Rauscher leukaemia virus (RLV) are poor stimulators of allogeneic and syngeneic T cells and express lower, but still significant, levels of MHC class II. In this paper we further investigated the mechanism of the dysfunction of DC. DC from infected animals did not cause anergy of T cells during coculture for 3 or 6 days. They did not release a substantial amount of soluble factors which could suppress T cell responses. The low T cell responses on stimulation using RLV-infected DC could be overcome by the addition of control DC. Pretreatment of these control DC with monoclonal antibody against MHC class II molecules completely blocked their ability to restore stimulation of T cells in the presence of infected DC. However, antibody against MHC class I or mismatched MHC class II molecules did not prevent restoration of function. The reduced labeling of surface MHC class II molecules previously reported was shown to reflect a loss in total class II molecules within the cells; MHC class I levels were unaltered by exposure to the virus. In DC from RLV-infected mice biosynthesis of MHC class II was decreased by around 50% at the transcriptional level in comparison with beta-actin. Thus, the down-regulation of surface class II molecules observed in DC following RLV infection is a consequence of a specific block in its biosynthesis and the failure of DC to stimulate T cells may be a direct consequence of the reduced class II levels. Since reduced stimulation by DC is also seen in HIV-1 infection in humans we speculate that a similar mechanism might operate in retroviral infection in man.

Animals↗
PubMed1996

Comparison between the phenotype and function of maturing dendritic cells from spleen and lymph nodes.

We compared the capacity of mature dendritic cells (DC) from lymph nodes and maturing DC from spleens in their capacity to stimulate responses to the small hapten picryl sulphonic acid (PIC) and to the same hapten conjugated to ovalbumin (PIC-OVA) and requiring processing. Surface expression of major histocompatibility complex (MHC) class II molecules, which are upregulated during maturation of splenic DC, were studied as an independent marker of maturation. Freshly isolated lymph node DC had a veiled appearance and high levels of class II expression. DC separated from suspensions of spleen cells expressed the DC-specific marker NLDC-145, but were small, had low levels of MHC class II molecules and expressed stem cell antigen. Those DC from spleen cells cultured for 24 and 48 hr showed the development of typical veiled DC morphology and high class II expression. Lymph node DC stimulated high levels of primary T-cell proliferation to PIC, but failed to stimulate primary responses to PIC-OVA. Splenic DC isolated immediately failed to stimulate primary responses to either antigen. More mature spleen DC stimulated responses both to PIC and PIC-OVA. Surprisingly, development of the capacity to stimulate responses to PIC preceded that of stimulating PIC-OVA responses. The capacity of the DC to process and present PIC-OVA was maintained during the culture period. The results indicate that both the form of the antigen and the source and maturity of the DC are critical in determining the responses stimulated in T lymphocytes.

Animals↗
PubMed1996

Antigen-presenting cells but not lymphocytes in the joint may indicate the cause of reactive arthritis.

T cells and antigen-presenting cells (APC) accumulate in the joint in reactive arthritis and there are reports that the T cells are a population selected for responsiveness to the causative agent. In this work, the latter view is questioned by detailed studies of the antigen specificities of the lymphocytes within the joint (SFMC) and peripheral blood (PBMC) of patients with reactive arthritis triggered by infection with Chlamydia trachomatis. Using a hanging-drop microculture system. SFMC displayed enhanced responses not only to antigens from the triggering organism, but also to other antigens, including PPD and tetanus toxoid, to which the patients were likely to have had prior exposure. No evidence was obtained for a dominant cross-reactive T-cell response to epitopes common to these antigen preparations, confirming the polyclonal nature of the infiltrate. In contrast to the broad specificity of the T-cell infiltrate, two experimental approaches indicated that APC within the joint carried chlamydial antigen. The failure of antigen-bearing APC to interact with T cells at this site may underlie the inability to clear microbial antigen from the joint.

Antigen Presentation↗