Human CD14hi monocytes and myeloid dendritic cells provide a cell contact-dependent costimulatory signal for early CD40 ligand expression.

CD40L on CD4(+) T cells plays a vital role in the activation of antigen-presenting cells, thus catalyzing a positive feedback loop for T-cell activation. Despite the pivotal juxtaposition of CD40L between antigen-presenting cells and T-cell activation, only a T-cell receptor stimulus is thought to be required for early CD40L surface expression. We show, for the first time, that CD40L expression on peripheral blood CD4(+) T cells is highly dependent on a cell-cell interaction with CD14(hi)CD16(-) monocytes. Interactions with ICAM-1, LFA-3, and to a lesser extent CD80/CD86 contribute to this enhancement of CD40L expression but are not themselves sufficient. The contact-mediated increase in CD40L expression is dependent on new mRNA and protein synthesis. Circulating myeloid dendritic cells also possess this costimulatory activity. By contrast, CD14(lo)CD16(+) monocytes, plasmacytoid dendritic cells, B-cell lymphoma lines, and resting, activated, and Epstein-Barr virus-immortalized primary B cells all lack the capacity to up-regulate early CD40L. The latter indicates that a human B cell cannot activate its cognate T cell to deliver CD40L-mediated help. This finding has functional implications for the role of biphasic CD40L expression, suggesting that the early phase is associated with antigen-presenting cell activation, whereas the late phase is related to B-cell activation.

Immunization with DNA topoisomerase I induces autoimmune responses but not scleroderma-like pathologies in mice.

OBJECTIVE: Anti-DNA topoisomerase I (anti-topo-I) antibody is a marker of systemic sclerosis (SSc). Anti-topo-I antibody levels are positively correlated with both disease severity and activity. However, its pathogenic role in SSc remains unclear. We investigated whether induction of an autoreactive antibody response is directly pathogenic in mice. METHODS: Autoimmune responses were induced in mice immunized with human recombinant topo-I (rhutopo-I). Both humoral and T cell-mediated autoimmune responses were assessed. Necropsy analyses were performed to determine pathologic changes in immunized mice. RESULTS: Autoimmune prone SJL and non-obese diabetic mice developed higher humoral autoreactive responses against mouse topo-I than did BALB/c and C56BL/6 mice. Splenic T cells also showed proliferative responses and interferon-gamma secretion in response to rhutopo-I. However, serum anti-topo-I antibody levels declined 2 months after the initial immunization. Neither weight loss nor dermal thickening was observed in mice during a followup period of 9 months. Whole-body necropsy analyses, including skin, lung, heart, kidney, gastrointestinal tract, and joints, showed no typical findings of human SSc. Coadministration of anti-CD25 and anti-CTLA-4 antibody with the initial immunization resulted in higher titers of anti-topo-I antibody, but these mice also did not develop SSc-like pathologic features. Development of an anti-topo-I response was not associated with acceleration of the recognized abnormalities in tight-skin mice. CONCLUSION: Although tolerance was broken and anti-topo-I antibody was induced by immunization with rhutopo-I in mice, induction of this antibody was not sufficient to induce SSc-like disease.

T cell lines from systemic sclerosis patients and healthy controls recognize multiple epitopes on DNA topoisomerase I.

Anti-DNA topoisomerase I (topo-I) antibodies are exclusively detected in patients with systemic sclerosis (SSc). Participation of this topo-I-specific autoimmune response in the pathogenesis of SSc has been actively investigated, but remains unproven. Here we characterized the peripheral T cell proliferative response to recombinant topo-I (rtopo-I) in 16 SSc patients with circulating anti-topo-I antibody. A low level (cpm < 2000) T cell proliferation (SI > 3) was detected in 6 (38%) of 16 patients. This low level response was similar to those previously observed in healthy controls. We established 56 topo-I-specific T cell lines recognizing 13 distinct T cell epitopes on topo-I from 4 SSc patients and 2 healthy controls. These T cell lines were established from in vitro activated PBMC (CD25(+)) by rtopo-I antigen. However they did not have the phenotype of regulatory T cells. Notably, 40 (71%) of the 56 T cell lines recognizing a common epitope were established from one patient. DNA sequencing of the T cell receptor cDNA produced an identical sequence indicating these T cells were from a single topo-I-specific T cell precursor. These results suggest that topo-I-specific T cells can become clonally expanded in some patients and may contribute to the pathogenesis of this disease.

Molecular recognition patterns of serum anti-DNA topoisomerase I antibody in systemic sclerosis.

Autoreactive anti-DNA topoisomerase I (anti-Topo I) Abs are commonly detected in sera of systemic sclerosis (SSc) patients. Our studies have established a positive correlation between the levels of serum anti-Topo I Abs and both disease severity and activity of SSc. The molecular targets of anti-Topo I Ab on Topo I domains remain to be further defined. In this report, we studied the molecular recognition pattern of serum anti-Topo I Ab in 52 SSc patients. The highest reactivity of serum anti-Topo I Abs was against the core subdomains I and II (aa 207-441) and, to a lesser extent, against the core subdomain III (aa 433-636) of Topo I. The linker domain (aa 636-712) and the C-terminal domain (aa 713-765) had much less reactivity than the core domain (aa 207-636). Strikingly, very little reactivity was directed against the N-terminal domain (aa 1-213) by serum anti-Topo I Ab. This molecular recognition pattern was consistent among all SSc serum samples studied. Results from patients with serial serum samples indicated that this pattern remained unchanged over time. Interestingly, some naive B cells from healthy controls, upon transformation by EBV, produced IgM Abs against Topo I. These Abs had low affinity for Topo I and reacted equally to all domains of Topo I. The molecular recognition pattern of serum anti-Topo I Ab in SSc suggests the presence of a unique antigenic stimulation in vivo in this disease.

Escherichia coli expressing recombinant antigen and listeriolysin O stimulate class I-restricted CD8+ T cells following uptake by human APC.

Vaccination against cancer or intracellular pathogens requires stimulation of class I-restricted CD8(+) T cells. It is therefore important to develop Ag delivery vectors that will promote cross-presentation by APCs and stimulate appropriate inflammatory responses. Toward this goal, we tested the potential of Escherichia coli as an Ag delivery vector in in vitro human culture. Bacteria expressing enhanced green fluorescent protein were internalized efficiently by dendritic cells, as shown by flow cytometry and fluorescence microscopy. Phenotypic changes in DC were observed, including up-regulation of costimulatory molecules and IL-12p40 production. We tested whether bacteria expressing recombinant Ags could stimulate human T cells using the influenza matrix protein as a model Ag. Specific responses against an immunodominant epitope were seen using IFN-gamma ELISPOT assays when the matrix protein was coexpressed with listeriolysin O, but not when expressed alone. THP-1 macrophages were also capable of stimulating T cells after uptake of bacteria, but showed slower kinetics and lower overall levels of T cell stimulation than dendritic cells. Increased phagocytosis of bacteria induced by differentiation of THP-1 increased their ability to stimulate T cells, as did opsonization. Presentation was blocked by proteasome inhibitors, but not by lysosomal protease inhibitors leupeptin and E64. These results demonstrate that recombinant E. coli can be engineered to direct Ags to the cytosol of human phagocytic APCs, and suggest possible vaccine strategies for generating CD8(+) T cell responses against pathogens or tumors.

Rapid and extensive membrane reorganization by dendritic cells following exposure to bacteria revealed by high-resolution imaging.

Using live cell imaging, we demonstrate that immature dendritic cells (DC) derived from human peripheral blood monocytes undergo pronounced morphologic changes in vitro within minutes of exposure to unopsonized Escherichia coli, developing extensive membrane veils that efficiently capture additional bacteria. Internalization does not occur in the veils, but instead, bacteria are transported to the central region of the cell, where they sink directly into the plasma membrane. In contrast, exposure to polystyrene beads does not induce notable changes in cell morphology, and DC do not efficiently capture beads when introduced alone or mixed with bacteria. Long dendritic processes were also visualized in some cells that allowed capture of clumps of bacteria at a distance of more than 100 microm. These results demonstrate that immature DC can distinguish between inert particles and bacteria and alter their shape and phagocytic capacity in response to the latter.

Correlation of serum anti-DNA topoisomerase I antibody levels with disease severity and activity in systemic sclerosis.

OBJECTIVE: To investigate correlations between serum levels of topoisomerase I-specific antibody (anti-topo I) and clinical features of systemic sclerosis (SSc), including disease severity (the total skin score [TSS]) and disease activity. METHODS: Using highly sensitive enzyme-linked immunosorbent assays, we measured the levels of anti-topo I antibody, including total IgG, individual IgG subclasses, and IgA, and analyzed their correlations with the TSS in 59 patients with SSc, all of whom had diffuse cutaneous involvement. Serial serum samples were obtained from 11 of these patients. RESULTS: The titers of anti-topo I antibody, including IgG and IgA, were positively correlated with the TSS, a measure of SSc disease severity. In 8 of the 11 patients from whom serial serum samples were obtained, changes in the levels of both IgG and IgA, when detectable, paralleled changes in the TSS. In 3 patients, an increasing anti-topo I IgG level preceded an increase in the TSS. The level of each IgG subclass also correlated with and tended to parallel the TSS. The patients with very active disease had higher mean IgG (P < 0.001) and IgA (P < 0.05) titers than did those with inactive disease. CONCLUSION: Serum levels of anti-topo I antibody correlate positively with disease severity and disease activity in SSc.

The male silkworm moth (Antheraea pernyi) is a key ingredient in hu-bao and sheng-bao for specific prolongation of the life-span of the male fruit fly (Drosophila melanogaster).

It is well established in Traditional Chinese Medicine that certain natural products, such as male silkworm moths, have different therapeutic effects on men than on women. These natural products have been used as dietary supplements specifically formulated for men or for women. However, this presumed sex-specific effect of certain natural products has not yet been confirmed experimentally with animal models or in human clinical trials. Here, using the fruit fly (Drosophila melanogaster) as a longevity model, we examined the effect of hu-bao (HB) and seng-bao (SB), two marketed health products made from a mixture of natural ingredients. Our results convincingly demonstrate that the effect of HB and SB are indeed specific for the male fly. The life-span of the male was significantly increased when HB or SB was added to the culture medium. In contrast, neither HB nor SB had much effect on the female fly. Upon removal of the male silkworm moth ingredient from HB or SB, the life-span prolongation effect of HB and SB was drastically diminished. Only with the addition of the male silkworm moth did the culture medium show a statistically significant life-span prolongation effect. This result suggests that the male silkworm moth is a key ingredient, in combination with other components, for specific prolongation of the life-span of male flies.