The precise mechanisms underlying anti-inflammatory effects of intravenous immunoglobulin (IVIg) therapies remain elusive. The sialylated IgG fraction within IVIg has been shown to be therapeutically more active in mouse models. Functionally, it has been suggested that IgG undergoes conformational changes upon Fc-sialylation which sterically impede binding to conventional FcγRs, but simultaneously allow binding to human DC-SIGN (SIGN-R1 in mice) and also CD23. These latter C-type lectins have been proposed responsible for the immunomodulatory effects in mouse models. However, there is conflicting evidence supporting direct interactions between sialylated human IgG and CD23/DC-SIGN. While cells expressing human CD23 and DC-SIGN in their native configuration bound their natural ligands IgE and ICAM-3, respectively, no IgG binding was observed, regardless of Fc-glycan sialylation in any context (with or without bisection and/or fucosylation) or presence of sialylated Fab-glycans. This was tested by both by FACS and a novel cellular Surface Plasmon Resonance imaging (cSPRi) approach allowing for monitoring low-affinity but high-avidity interactions. In summary, we find no evidence for human CD23 or DC-SIGN being bona fide receptors to human IgG, regardless of IgG Fc- or Fab-glycosylation status. However, these results do not exclude the possibility that either IgG glycosylation or C-type lectins affect IVIg therapies.

Chronic lymphocytic leukaemia (CLL) cells encounter T-cells and proliferate in response to T-cell signals in the lymph node microenvironment. In this report we determined interleukin 21 (IL21) function in CLL and showed that IL21 and interleukin 4 (IL4) act co-operatively to promote leukaemic cell proliferation without apoptosis or differentiation We further show that IL21 increased side population (SP) cells, which are associated with resistance to chemotherapy and increased self-renewal capacity in CLL. IL21 and IL4 are the major cytokines produced by the recently described CD4(+) T follicular helper (Tfh) cell subset. Determination of Tfh cells in peripheral blood showed that patients had significantly increased numbers as compared to normal subjects although no association was found between Tfh numbers and IGHV gene mutational status or clinical stage. Our data suggests that the Tfh cytokines, IL4 and IL21, contribute to driving leukaemic cell proliferation in the lymph node microenvironment, and may contribute to the specific production of cells resistant to conventional chemotherapy. We suggest that increased circulating Tfh cells is a component of T-cell dysregulation in CLL. Our findings have implications for the therapeutic use of IL21.
© 2013 John Wiley & Sons Ltd.

One characteristic of chronic lymphocytic leukaemia (CLL) lymphocytes is high expression of CD23, which has previously been identified as a downstream target for NOTCH2 signalling. The mechanisms regulating NOTCH2-dependent CD23 expression, however, are largely unknown. This study showed that peripheral CLL cells overexpressed transcriptionally active NOTCH2 (N2(IC)), irrespective of their prognostic marker profile. When placed in culture, NOTCH2 activity was spontaneously decreased in 25 out of 31 CLL cases (81%) within 24 h. DNA-bound N2(IC) complexes could be maintained by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or by gamma-interferon (IFN-gamma), two CLL characteristic inducers of CD23 expression. Inhibition of PKC-delta by RNA interference or by rottlerin antagonised PMA-induced NOTCH2 activation and also suppressed NOTCH2 activity in CLL cases with constitutively activated NOTCH2 signalling. In 23 out of 29 CLL cases tested (79%), DNA-bound N2(IC) complexes were found to be resistant to the gamma-secretase inhibitor (GSI) DAPT, suggesting that GSIs will be only effective in a subset of CLL cases. These data suggest that deregulation of NOTCH2 signalling is critically involved in maintaining the malignant phenotype of CLL lymphocytes and point to a link between PKC-delta and NOTCH2 signalling in the leukemic cells.

Airway smooth muscle cells play a key role in remodeling that contributes to airway hyperreactivity. Airway smooth muscle remodeling includes hypertrophy and hyperplasia. It has been previously shown that the expression of CD23 on ASMC in rabbits can be induced by the IgE component of the atopic serum. We examined if other components of atopic serum are capable of inducing CD23 expression independent of IgE.
Serum starved huASMC were stimulated with either IL-4, GM-CSF, IL-13, IL-5, PGD2, LTD4, tryptase or a combination of IL-4, IL-5, IL-13 each with GM-CSF for a period of 24 h. CD23 expression was analyzed by flow cytometry, western blot, and indirect immunofluorescence.
The CD23 protein expression was upregulated in huASMC in response to IL-4, GM-CSF, and IL-4/GM-CSF. The percentage of cells with increased fluorescence intensity above the control was 25.1 +/- 4.2% (IL-4), 15.6 +/- 2.7% (GM-CSF) and 32.9 +/- 13.9% (IL-4/GMCSF combination)(n = 3). The protein content of IL-4/GMCSF stimulated cells was significantly elevated. Expression of CD23 in response to IL-4, GM-CSF, IL-4/GM-CSF was accompanied by changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling. Western blot revealed abundant expression of the IL-4Ralpha and a low level expression of IL-2Rgammac in huASMC. Stimulation with IL-4 resulted in the phosphorylation of STAT-6 and an increase in the expression of the IL-2Rgammac.
CD23 on huASMC is upregulated by IL-4, GM-CSF, and IL-4/GM-CSF. The expression of CD23 is accompanied by an increase in cell volume and an increase in protein content per cell, suggesting hypertrophy. Upregulation of CD23 by IL-4/GM-CSF results in phenotypic changes in huASMC that could play a role in cell migration or a change in the synthetic function of the cells. Upregulation of CD23 in huASMC by IL-4 and GM-CSF can contribute to changes in huASMC and may provide an avenue for new therapeutic options in asthma targeting ASMC.