Dendritic cells from control but not atopic donors respond to contact and respiratory sensitizer treatment in vitro with differential cytokine production and altered stimulatory capacity.

BACKGROUND: Chemical haptens induce both contact and allergic respiratory disease with dendritic cells (DCs) controlling and directing immune responses in vivo. Contact and respiratory haptens may promote differential cytokine production yet distinguishing these effects in vitro remains difficult due to human donor variability. Objective We sought to determine the effect of atopic status on the ability of DC to respond to contact and respiratory sensitizer treatment in vitro as DC from atopic donors are believed to promote Th2-type responses. METHODS: Enriched DC from control or atopic donors were treated for 4 h with levels of the contact sensitizer 2,4-dinitrochlorobenzene (DNCB) or the respiratory sensitizer trimellitic anhydride (TMA) that did not reduce cell viability. A sensitive intracellular detection technique was used to measure cytokine production, while T cell responses were assessed in a mixed leucocyte reaction. RESULTS: DC from control, non-atopic, donors produced cytokines differentially in response to sensitizer treatment; DNCB treatment significantly increased the production of Th1 cytokines IL-12 and IFN-gamma while TMA induced the production of IL-13. Control donor DC treated with TMA stimulated less in a mixed leucocyte reaction than untreated cells with any response reduced further by blocking IL-13 in culture. However, DC from atopic donors showed no significant alteration in either cytokine production or T cell stimulatory capacity after sensitizer treatment. CONCLUSION: Haptens modulate DC by changing the production of cytokines that may play a role in T cell stimulation and subsequent polarization of the immune response. DC from atopic donors were unresponsive to chemical sensitizer treatment, and may be deficient in inducing divergent T cell responses.

The fraction 1 and V protein antigens of Yersinia pestis activate dendritic cells to induce primary T cell responses.

The F1 and V antigens of Yersinia pestis, despite acting as virulence factors secreted by the organism during infection, also combine to produce an effective recombinant vaccine against plague, currently in clinical trial. The protective mechanisms induced by rF1 + rV probably involve interactions with dendritic cells (DC) as antigen uptake, processing and presenting cells. To study such interactions, naive ex vivo DC from bone marrow, spleen and lymph node were cultured with rF1, rV or combined antigens and demonstrated to secrete interleukin (IL)-4 and IL-12 into the culture supernatant. Cytokine production in response to pulsing was dependent on the maturity of the bone marrow-derived DC culture, so that pulsed 8-day-old cultures had accumulated significantly more intracellular IL-4 and IL-12 than unpulsed cells. DC, pulsed with rF1 + rV for 2-24 h, were able to prime naive autologous lymph node T cells to proliferate in an antigen dose-dependent manner, with an order of potency of 3d bone marrow-derived DC (BMDC) > 7d BMDC > splenic DC. Significantly, cell-free supernatants from rF1 + rV-pulsed BMDC and splenic DC were also able to induce specific primary responses effectively in naive T cells, suggesting that these supernatants contained stimulatory factor(s). This study suggests an important role for DC, or factors secreted by them, in the induction of protective immunity to plague by the rF1 and rV antigens.

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.

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.

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.

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.

Effects of murine leukemia viruses on the function of dendritic cells.

In asymptomatic human immunodeficiency virus-1 infection T cells respond normally to allogeneic dendritic cells (DC), but DC show reduced stimulatory capacity. By contrast in HTLV-1 infection no significant changes in allogeneic stimulation were seen but DC-stimulated activity of autologous T cells. In seeking animal models relevant to these diseases the effects of two murine leukemia retroviruses, Rauscher leukemia virus (RLV) and Moloney leukemia virus (MLV) on the function of dendritic cells and T cells in a primary mixed leucocyte reaction have been tested. Treatment by RLV in vitro suppressed the ability of DC to stimulate allogeneic T cells from healthy animals. MLV at the same concentration did not significantly affect the ability of DC to stimulate allogeneic T cells, but provoked considerable enhancement of the low level stimulation by DC in the syngeneic system. Similar results were obtained following in vivo exposure to viruses. Two pieces of evidence suggested that these effects were due to impairment of DC function and were not operating through infection of T cells. Firstly, exposure of T cells directly to virus in vitro and in vivo before stimulation with untreated allogeneic DC caused no significant alteration in T cell activity. Secondly, the impact of murine leukemia virus on DC function was not abrogated when infected DC were added to normal T cells and cultured in the presence of zidovudine. Treatment of DC by RLV caused a decrease of cluster formation with allogeneic T cells. No statistically significant influence of MLV was observed on cluster formation after 3-h of incubation in the allogeneic system. However, after 18-h incubation MLV-treated DC formed fewer clusters with T cells than untreated DC. At the same time a stimulatory effect of MLV on DC cluster formation with syngeneic T cells was found. Considerable decrease was found in major histocompatibility complex class II antigen and LFA-1 receptor expression on the DC surface in mice infected by RLV. MLV induced no significant changes. These mouse retroviruses can therefore cause changes in DC function similar to those already reported using human retroviruses and may provide models for studying their effects.

Increased frequency of DR4 in systemic onset juvenile chronic arthritis.

Eighty-one patients with systemic onset juvenile chronic arthritis (JCA) have been tested for HLA-A, -B, -C, and -DR antigens. This study confirms previous reports of an increased incidence of DR4 in these patients. Subdivision of the patients according to their disease course over ten years showed different HLA associations with different disease courses. The frequency of DR4 tended to be greater in patients with less severe disease. There was also an increased incidence of HLA B27 in patients in whom relapses of disease were associated with intercurrent infections.

The effect of retinoids on dendritic cell function.

The effect of retinoid administration on the antigen presenting function of mouse dendritic cells (DC) was assessed. Culturing spleen cells with retinoic acid (10(-10)-10(-4) M) had no effect on the numbers of DC separated from these cultures. However, DC isolated from retinoic-acid-treated cultures were less stimulatory than DC from untreated cultures when added to allogeneic lymphocytes in a mixed leucocyte culture. Allogeneic stimulation by DC was also inhibited by pulsing separated DC with retinoic acid (10(-6)-10(-4) M) for 2 h. Pulsing DC with lower doses of retinoic acid (10(-14)-10(-20) M) enhanced this response. DC isolated from animals maintained on VAA-enriched diets had a reduced capacity to stimulate allogeneic lymphocytes. The response of unseparated lymph node cells pulsed with retinoic acid to untreated allogeneic DC was inhibited by 10(-10)-10(-4) M retinoic acid but enhanced by lower doses (10(-14) M). However, the inhibitory effect of retinoids on the function of responding lymphoid populations was abolished on removal of DC from responding cells. The results indicate that immunomodulation by retinoids could occur via an effect on the efficiency of antigen presentation by DC.

Induction of autoimmunity with dendritic cells: studies on thyroiditis in mice.

The initiation and maintenance of thyroid autoimmunity by professional antigen-presenting cells were assessed by observing thyroiditis and induction of IgG antibodies to thyroglobulin (Tg). Dendritic cells (DC) were purified from spleens of CBA mice and T cells removed with anti-Thy 1 and complement. Some DC were pulsed with 25-500 micrograms/ml of mouse Tg in vitro and normal syngeneic mice received injections of 10(5) cells intravenously. In untreated animals only 1 thyroid out of 40 showed a lymphocyte infiltrate and antibody to Tg was rarely seen. In animals receiving normal DC without Tg, lymphocyte infiltration was seen 2-6 weeks later in 5 out of 33 thyroids and some animals produced low levels of antibody to thyroglobulin (8 of 33 animals). DC pulsed with 500 micrograms Tg/ml in vitro caused thyroid infiltration in 6 out of 15 animals but did not increase the incidence of anti-Tg antibodies. Lower doses had no effect. When 10(5) DC were given from animals with experimental allergic thyroiditis (EAT, induced with Tg in complete Freund's adjuvant, CFA) more than half of the recipient animals showed thyroiditis (8 out of 15) and autoantibody production (12 of 15 animals). DC may therefore play a role in the initiation and maintenance of autoimmunity by providing a stimulus for antigen-specific T cells.

HLA antigen frequencies among patients with juvenile chronic arthritis and amyloidosis: a brief report.

Amyloidosis is seen in a small number of patients with juvenile chronic arthritis (JCA). In order to determine whether particular HLA markers might predispose to the development of amyloid in JCA a group of 45 patients with amyloidosis confirmed by biopsy was typed for the HLA-A, B, C and DR loci. The results confirmed previous smaller studies that no HLA antigen detected by standard serological techniques was associated specifically with the development of amyloidosis. Those antigens which showed an altered frequency (ie. DR4 and DRw8) were known to be associated with the different types of JCA onset represented in this group.

Genetic susceptibility to early onset pauciarticular juvenile chronic arthritis: a study of HLA and complement markers in 158 British patients.

To investigate the genetics of susceptibility to early onset pauciarticular juvenile chronic arthritis (JCA), 158 unrelated ethnic British patients with a mean disease onset of 3.2 years, together with controls, were tested for HLA-A, B, C, and DR antigens. Additionally, 117 patients were also investigated for complement Bf and C4 markers. New observations included an increased frequency of the C4B 2 allotype (p corrected (pc) less than 0.02) and C4A 4,B 2 phenotype (p less than 0.0005). Findings suggested a unique increase of the haplotype HLA-DRw8, Bf*S, C4A*4, C4B*2, HLA-B39, possibly predisposing to more severe disease. Strong positive associations were confirmed with HLA antigens A2 (pc = 2.5 X 10(-8)), DRw8 (pc = 3.5 X 10(-14)), DR5 (pc less than 0.02), DRw52 (pc = 2.8 X 10(-6)) and DR5, w8 phenotype (pc = 3.9 X 10(-6)), and negative associations with DR7 (pc = 5.8 X 10(-7)), DR4 (pc less than 0.002), and DRw53 (pc = 0.004). Antinuclear antibody (ANA) seropositivity correlated with DR5 (p less than 0.02), and in children with chronic iridocyclitis (CIR) Bw62 incidence was raised (p less than 0.03) and B44 reduced (p less than 0.03). HLA-A2 was found in 88% of ANA+, CIR+ patients (p less than 0.01). A significant excess of DR5, w8 heterozygotes was present (relative risk = 41.1) and a lack of corresponding homozygotes. Results are inconsistent with a recessive, dominant, or intermediate mode of inheritance of susceptibility, and favour the existence of at least two DR linked 'disease' genes. Moreover, there may be an interaction in heterozygotes of combinatorial factors associated with DR5 and DRw8 in enhancing susceptibility. Possible immunogenetic mechanisms underlying the observed associations with three antigen classes are discussed. Evidence here suggests a role for the HLA-DQ locus in determining susceptibility to this disease.

HLA antigens in coeliac disease associated with malignancy.

Coeliac patients are at greater risk than the general population of developing malignant neoplasms, particularly lymphomas. The establishment at the Clinical Research Centre of a national collaborative study of coeliac patients with malignancy provided the opportunity to carry out HLA typing for 55 HLA-A, B and C and the 10 recognised DR antigens on a group of coeliac patients with malignancy. Study of a sample of 44 patients with biopsy proven coeliac disease and histologically confirmed malignancy, including 12 with malignant histiocytosis, and 57 coeliac patients without malignancy, failed to show any significant differences in antigen frequencies between patients with and without malignancy. These results indicate that there are no HLA genetic markers associated specifically with the development of malignancy in coeliac disease.

The preparation of plasma fibronectin antigen and antiserum.

Affinity chromatography of human plasma, using either gelatin or fibrinogen coupled to Sepharose 4B, depletes the plasma of fibronectin. Other 'contaminant' proteins are also bound by this procedure on the column and elute with the fibronectin, as shown by using the bound and eluted fraction to immunise rabbits. Such antisera can be rendered specific for fibronectin by absorption with fibronectin depleted plasma. Alternatively, purified fibronectin can be obtained by a two-stage chromatographic procedure using a second separation on Sephacryl S300, and immunization with this as antigen produces monospecific antiserum. This reacts both with plasma and tissue fibronectin.