ALBERT

Description: The CD150 receptor is expressed on activated T and B lymphocytes, dendritic cells, and monocytes. A TxYxxV/I motif in the CD150 cytoplasmic tail can bind different SH2-containing molecules, including tyrosine and inositol phosphatases, Src family kinases, and adaptor molecules. To analyze CD150-initiated signal transduction pathways, we used DT40 B-cell sublines deficient in these molecules. CD150 ligation on DT40 transfectants induced the extracellular signal-regulated kinase (ERK) pathway, which required SH2-containing inositol phosphatase (SHIP) but not SH2 domain protein 1A (SH2D1A). CD150-mediated Akt phosphorylation required Syk and SH2D1A, was negatively regulated by Lyn and Btk, but was SHIP independent. Lyn directly phosphorylated Y327 in CD150, but the Akt pathway did not depend on CD150 tyrosine phosphorylation and CD150-SHP-2 association. Analysis of CD150 and SH2D1A expression in non-Hodgkin and Hodgkin lymphomas revealed stages of B-cell differentiation where these molecules are expressed alone or coexpressed. Signaling studies in Hodgkin disease cell lines showed that CD150 is linked to the ERK and Akt pathways in neoplastic B cells. Our data support the hypothesis that CD150 and SH2D1A are coexpressed during a narrow window of B-cell maturation and SH2D1A may be involved in regulation of B-cell differentiation via switching of CD150-mediated signaling pathways. (Blood. 2004;104:4063-4070)

Description: Dendritic-cell (DC) migration to secondary lymphoid organs is crucial for the initiation of adaptive immune responses. Although LPS up-regulates CCR7 on DCs, a second signal is required to enable them to migrate toward the chemokine CCL19 (MIP-3β). We found that the nitric oxide (NO) donor NOR4 provides a signal allowing LPS-stimulated DCs to migrate toward CCL19. NO affects DC migration through both the initial activation of the cGMP/cGMP kinase (cGMP/cGK) pathway and a long-term effect that reduced cGK activity via negative feedback. Indeed, migration of DCs toward CCL19, unlike migration toward CXCL12 (SDF-1α), required inhibition of cGK. LPS increased both cGK expression and cGK activity as measured by phosphorylation of the key cGK target vasodilator-stimulated phosphoprotein (VASP). Because cGK phosphorylation of VASP can disrupt focal adhesions and inhibit cell migration, LPS-induced VASP phosphorylation may prevent DCs from migrating without a second signal. Long-term NOR4 treatment inhibited the increase in cGK-dependent VASP phosphorylation, releasing this brake so that DCs can migrate. NO has been implicated in the regulation of autoimmunity through its effect on T cells. Our results suggest that NO regulation of DC migration and cytokine production may contribute to the protective effects of NO in autoimmune disorders.

Description: Macrophages and dendritic cells play an important role in regulating B-cell responses, including proliferation to antigens such as trinitrophenyl (TNP)—Ficoll and TNP-Brucella abortus. However, the mechanisms and molecule(s) that regulate these processes are relatively undefined. In this report, we show that human macrophages generated in vitro strongly costimulate proliferation of dense human tonsillar B cells ligated via their B-cell antigen receptor (BCR) but not proliferation via CD40. Similarly, dendritic cells also markedly enhance BCR-activated B-cell proliferation. Soluble molecule(s) are required for human macrophages to costimulate proliferation of B cells triggered via their BCR. Importantly, a TACI (trans-membrane activator and CAML interactor)—Fc fusion protein inhibits both macrophage- and dendritic cell (DC)—dependent BCR-activated B-cell proliferation, indicating a requirement for at least one of the known TACI ligands, BAFF and/or APRIL. Consistent with a major role for BAFF, macrophages release BAFF at levels sufficient to potently costimulate BCR-induced B-cell proliferation. In addition, BAFF is more than 100-fold more potent than APRIL in enhancing BCR-mediated human B-cell proliferation. Furthermore, immunodepletion of APRIL under conditions that prevent APRIL-mediated B-cell costimulation does not block macrophage enhancement of B-cell proliferation. Finally, there is no correlation between the high levels of a proliferation-inducing ligand (APRIL) expressed by macrophages compared with DCs and the similar abilities of macrophages and DCs to enhance BCR-stimulated B-cell proliferation. In summary, our results suggest that macrophage- and DC-derived B-cell—activating factor belonging to the TNF family (BAFF) represents a key molecule by which macrophages and DCs directly regulate human B-cell proliferative responses to T-cell—independent stimuli.

Description: The effects of estrogen on the immune system are still largely unknown. We have investigated the effect of 17β-estradiol (E2) on human monocyte-derived immature dendritic cells (iDCs). Short-term culture in E2 had no effect on iDC survival or the expression of cell surface markers. However, E2 treatment significantly increased the secretion of interleukin 6 (IL-6) in iDCs and also increased secretion of osteoprotegerin (OPG) by DCs. Furthermore, E2 significantly increased secretion of the inflammatory chemokines IL-8 and monocyte chemoattractant protein 1 (MCP-1) by iDCs, but not the production of the constitutive chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). However, after E2 pretreatment the lipopolysaccharide (LPS)–induced production of MCP-1, TARC, and MDC by DCs was clearly enhanced. Moreover, mature DCs pretreated with E2 stimulated T cells better than control cells. Finally, we found that E2 provides an essential signal for migration of mature DCs toward CCL19/macrophage inflammatory protein 3β (MIP3β). In summary, E2 may affect DC regulation of T-cell and B-cell responses, as well as help to sustain inflammatory responses. This may explain, in part, the reason serum levels of estrogen correlate with the severity of certain autoimmune diseases.

Description: Introduction: B-CLL is characterised by the accumulation of long-lived CD5+ B cells. Mature B cells can be activated via TLRs to produce immunoglobulin and proliferate. We have previously reported differential surface expression of CD180, TLR-4 associated receptor, by B-CLL cells in relation to IgVh gene status (Porakishvili N et al., 2004; Blood (ASH annual meeting abstracts) 104: 2807). To further characterise subsets of leukemic cells, we assessed B-CLL cells for TLR mRNA expression. Method: B cells from untreated (n=24) and treated (CHOP n=3, Thalidomide n=1 and chlorambucil and prednisolone n=2) CLL patients, and healthy aged matched controls, (n=14) were isolated by CD19+ selection using iMACs columns (Miltenyi Biotec), total RNA extracted (Qiagen) and primers for TLR2, TLR4, TLR9, CD180 and the adaptor protein MYD88 were used for TaqMan PCR analysis (ABI). GAPDH was used for normalization. Data were expressed as mean delta cycle number (ct) ± standard deviation, giving a value inversely related to the level of expression of the specific gene. Patients were separated into groups according to IgVh gene mutation status or level of Zap 70 expression. Statistical analysis was performed using by Mann-Whitney U test and Spearman rank correlation or Pearson’s correlation coefficient. Results: Overall, there was a heterogeneous pattern of TLR expression in patients and controls with no significant differences between them. Treated patients were characterised by higher mRNA levels for the all TLRs measured (TLR9 p=0.018) and MYD88 (MYD88 p=0.026) compared with untreated patients, despite no significant difference of their GAPDH levels (p〉0.05). Significant correlations were seen between mRNA levels for the TLRs within the age matched control group (all p2%) or unmutated IgVh genes (unmutated

Description: Dendritic-cell (DC)-associated C-type lectin receptors (CLRs) take up antigens to present to T cells and regulate DC functions. DCAL-2 is a CLR with a cytosolic immunoreceptor tyrosine-based inhibitory motif (ITIM), which is restricted to immature DCs (iDCs), monocytes, and CD1a+ DCs. Cross-linking DCAL-2 on iDCs induced protein tyrosine phosphorylation and MAPK activation as well as receptor internalization. To test if DCAL-2 is involved in DC maturation and cytokine expression, we stimulated iDCs with anti-DCAL-2 mAb with or without LPS, zymosan, or CD40L. While anti-DCAL-2 did not induce iDCs to mature, it did up-regulate CCR7 expression and IL-6 and IL-10 production. DCAL-2 signals augmented DC maturation induced by LPS or zymosan, increasing both CCR7 and DC-LAMP expression. Of interest, DCAL-2 ligation had the opposite effects on TLR versus CD40L signaling: anti-DCAL-2 suppressed TLR-induced IL-12 expression, but significantly enhanced CD40L-induced IL-12 production. DCAL-2 ligation also suppressed the ability of TLR-matured DCs to induce IFN-γ-secreting Th1 cells but augmented the capacity of CD40L-matured DCs to polarize naive T cells into Th1 cells. Thus, DCAL-2 may program DCs differently depending on whether DCs are signaled via TLRs or by T cells. DCAL-2 may be a potential immunotherapeutic target for modulating autoimmune diseases or for developing vaccines.