Aaron J. Marshall

Osteoprotegerin, a Crucial Regulator of Bone Metabolism, Also Regulates B Cell Development and Function

Osteoprotegerin (OPG) is a CD40-regulated gene in B cells and dendritic cells (DCs). We investigated the role of OPG in the immune system by generating opg−/− mice. Like its role as a regulator of bone metabolism, OPG also influences processes in the immune system, notably in B cell development. Ex vivo, opg−/− pro-B cells have enhanced proliferation to IL-7, and in opg−/− spleen, there is an accumulation of type 1 transitional B cells. Furthermore, opg−/− bone marrow-derived DCs are more effective in stimulating allogeneic T cells than control DCs. When challenged with a T-dependent Ag, opg−/− mice had a compromised ability to sustain an IgG3 Ag-specific response. Thus, in the immune system, OPG regulates B cell maturation and development of efficient Ab responses.

PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity

The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.

TAPP1 and TAPP2 Are Targets of Phosphatidylinositol 3-Kinase Signaling in B Cells: Sustained Plasma Membrane Recruitment Triggered by the B-Cell Antigen Receptor

ABSTRACT We report the characterization of two signal transduction proteins related to Bam32, known as TAPP1 and TAPP2. Bam32, TAPP1, and TAPP2 share several characteristics, including small size (32 to 47 kDa), lack of enzymatic domains, high conservation between humans and mice, and the presence of pleckstrin homology (PH) domains near their C termini which contain the 3-phosphoinositide-binding motif. Unlike Bam32, the N-terminal regions of TAPP1 and TAPP2 contain a second PH domain. TAPP1 and TAPP2 transcripts are expressed in a variety of tissues including lymphoid tissues. Using live-cell imaging, we demonstrate that TAPP1 and TAPP2 are recruited to the plasma membrane of BJAB human B-lymphoma cells upon activation through the B-cell antigen receptor (BCR). The C-terminal PH domain is necessary and sufficient for BCR-induced membrane recruitment of both TAPP1 and TAPP2. Blockade of phosphatidylinositol 3-kinase (PI3K) activity completely abolished BCR-induced recruitment of TAPP1 and TAPP2, while expression of active PI3K is sufficient to drive constitutive membrane localization of TAPP1 and TAPP2. TAPP1 and TAPP2 preferentially accumulate within ruffled, F-actin-rich areas of plasma membrane, suggesting a potential role in PI3K-driven cytoskeletal reorganization. Like Bam32, BCR-driven TAPP1 and TAPP2 recruitment is a relatively slow and sustained response, in contrast to Btk recruitment and Ca2+ mobilization responses, which are rapid and transient. Consistent with recent studies indicating that Bam32, TAPP1, and TAPP2 can bind to PI(3,4)P2, we find that membrane recruitment correlates well with production of PI(3,4)P2 but not with that of PI(3,4,5)P3. Our results indicate that TAPP1 and TAPP2 are direct targets of PI3K signaling that are recruited into plasma membranes with distinctive delayed kinetics and accumulate within F-actin-rich membrane ruffles. We postulate that the TAPPs function to orchestrate cellular responses during the sustained phase of signaling.

Role of the phosphoinositide 3‐kinase p110δ in generation of type 2 cytokine responses and allergic airway inflammation

Phosphoinositide 3‐kinases (PI3K) regulate immune activation via their roles in signal transduction of multiple classes of receptors. Here, we examined the effect of genetic inactivation of the hemopoietic cell‐restricted PI3K isoform p110δ on systemic cytokine and chemokine responses and allergic airway inflammation. We found that type 2 cytokine responses (IL‐4, IL‐5 and IL‐13) are significantly decreased in p110δ mutants, whereas type 1 cytokine responses (IFN‐γ and CXCL10) were robust. Elevated IFN‐γ production during the primary response to ovalbumin (OVA) was associated with reduced production of the regulatory cytokine IL‐10. IFN‐γ and IL‐10 production normalized after secondary OVA immunization; however, type 2 cytokine production was persistently reduced. Type 2 cytokine‐dependent airway inflammation elicited by intranasal challenge with OVA was dramatically reduced, with reduced levels of eosinophil recruitment and mucus production observed in the lungs. Induction of respiratory hyper‐responsiveness to inhaled methacholine, a hallmark of asthma, was markedly attenuated in p110δ‐inactivated mice. Adoptive transfer of OVA‐primed splenocytes from normal but not p110δ‐inactivated mice could induce airway eosinophilia in naive, airway‐challenged recipient mice. These data demonstrate a novel functional role for p110δ signaling in induction of type 2 responses in vivo and may offer a new therapeutic target for Th2‐mediated airway disease.

The p110δ Isoform of Phosphatidylinositol 3-Kinase Controls Susceptibility to Leishmania major by Regulating Expansion and Tissue Homing of Regulatory T Cells

Resistance to Leishmania major and most intracellular pathogens is usually associated with a strong T cell-mediated immunity, particularly a CD4+ Th1 response. Mice with an inactivating knock-in mutation in the p110δ isoform of PI3K (referred to as p110δD910A) show severely impaired T cell responses. Because a strong T cell response is thought to mediate resistance to intracellular pathogens, we examined the outcome of L. major infection in p110δD910A mice. Paradoxically, p110δD910A mice on “resistant” and “susceptible” genetic backgrounds showed more robust resistance manifested as significantly reduced lesion size and accelerated parasite clearance. This enhanced resistance was associated with dramatically diminished immune responses, including impaired cell proliferation and effector cytokine (IFN-γ and TNF) production. Interestingly, the ability of macrophages and dendritic cells from p110δD910A mice to produce NO and destroy Leishmania parasites was similar to those of wild-type mice. We show that the enhanced resistance of p110δD910A mice was due to impaired expansion and effector functions of regulatory T cells (Tregs). Adoptive transfer studies demonstrated that p110δD910A mice lost their increased resistance when given enriched Tregs from wild-type mice. We suggest on the basis of these and further observations that the lack of this enzyme prominently affects Treg expansion and homing to infection sites, and that in the absence of Tregs, weak Th1 responses are capable of containing parasites and prevent pathology. We also suggest that temporary pharmacological inhibition of this enzyme may be a very effective form of treatment against cutaneous leishmaniasis.

Dendritic Cell-Associated Lectin-1: A Novel Dendritic Cell-Associated, C-Type Lectin-Like Molecule Enhances T Cell Secretion of IL-4

We have characterized dendritic cell (DC)-associated lectin-1 (DCAL-1), a novel, type II, transmembrane, C-type lectin-like protein. DCAL-1 has restricted expression in hemopoietic cells, in particular, DCs and B cells, but T cells and monocytes do not express it. The DCAL-1 locus is within a cluster of C-type lectin-like loci on human chromosome 12p12–13 just 3′ to the CD69 locus. The consensus sequence of the DCAL-1 gene was confirmed by RACE-PCR; however, based on sequence alignment with genomic DNA and with various human expressed sequence tags, we predict that DCAL-1 has two splice variants. C-type lectins share a common sequence motif of 14 invariable and 18 highly conserved aa residues known as the carbohydrate recognition domain. DCAL-1, however, is missing three of the cysteine residues required to form the standard carbohydrate recognition domain. DCAL-1 mRNA and protein expression are increased upon the differentiation of monocytes to CD1a+ DCs. B cells also express high levels of DCAL-1 on their cell surface. Using a DCAL-1 fusion protein we identified a population of CD4+ CD45RA+ T cells that express DCAL-1 ligand. Coincubation with soluble DCAL-1 enhanced the proliferation of CD4+ T cells in response to CD3 ligation and significantly increased IL-4 secretion. In contrast, coincubation with soluble DC-specific ICAM-3-grabbing nonintegrin (CD209) fusion protein as a control had no effect on CD4+ T cell proliferation or IL-4 and IFN-γ secretion. Therefore, the function of DCAL-1 on DCs and B cells may act as a T cell costimulatory molecule, which skews CD4+ T cells toward a Th2 response by enhancing their secretion of IL-4.

The B Lymphocyte Adaptor Molecule of 32 Kilodaltons (Bam32) Regulates B Cell Antigen Receptor Internalization

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is an adaptor that plays an indispensable role in BCR signaling. In this study, we found that upon BCR ligation, Bam32 is recruited to the plasma membrane where it associates with BCR complexes and redistributes and internalizes with BCRs. BCR ligation induced colocalization of Bam32 with lipid rafts, clathrin, and actin filaments. An inhibitor of Src family protein tyrosine kinases (PTKs) blocked both BCR-induced tyrosine phosphorylation of Bam32 and BCR internalization. Moreover, BCR internalization is impaired in Bam32−/− and Lyn−/− cells, and expression of Bam32 with a mutation of its tyrosine phosphorylation site (Y139F) inhibited BCR internalization. These data suggest that Bam32 functions downstream of Src family PTKs to regulate BCR internalization. Bam32 deficiency does not affect tyrosine phosphorylation of clathrin or the association of clathrin with lipid rafts upon BCR cross-linking. However, BCR-induced actin polymerization is impaired in Bam32−/− cells. Collectively, these findings indicate a novel role of Bam32 in connecting Src family PTKs to BCR internalization by an actin-dependent mechanism.

FDC-SP, a novel secreted protein expressed by follicular dendritic cells

To define better the molecular basis for follicular dendritic cell (FDC) function, we used PCR-based cDNA subtraction to identify genes specifically expressed in primary FDC isolated from human tonsils. In this work we report the discovery of a novel gene encoding a small secreted protein, which we term FDC-SP (FDC secreted protein). The FDC-SP gene lies on chromosome 4q13 adjacent to clusters of proline-rich salivary peptides and C-X-C chemokines. Human and mouse FDC-SP proteins are structurally unique and contain a conserved N-terminal charged region adjacent to the leader peptide. FDC-SP has a very restricted tissue distribution and is expressed by activated FDCs from tonsils and TNF-α-activated FDC-like cell lines, but not by B cell lines, primary germinal center B cells, or anti-CD40 plus IL-4-activated B cells. Strikingly, FDC-SP is highly expressed in germinal center light zone, a pattern consistent with expression by FDC. In addition, FDC-SP is expressed in leukocyte-infiltrated tonsil crypts and by LPS- or Staphylococcus aureus Cowan strain 1-activated leukocytes, suggesting that FDC-SP can also be produced in response to innate immunity signals. We provide evidence that FDC-SP is posttranslationally modified and secreted and can bind to the surface of B lymphoma cells, but not T lymphoma cells, consistent with a function as a secreted mediator acting upon B cells. Furthermore, we find that binding of FDC-SP to primary human B cells is markedly enhanced upon activation with the T-dependent activation signals such as anti-CD40 plus IL-4. Together our data identify FDC-SP as a unique secreted peptide with a distinctive expression pattern within the immune system and the ability to specifically bind to activated B cells.

Role of phosphoinositide 3-kinase p110δ in TLR4- and TLR9-mediated B cell cytokine production and differentiation

Phosphoinositide 3-kinase (PI3K) enzymes play key roles in signaling via antigen receptors and cytokine receptors and isoform-selective PI3K inhibitors are being evaluated as targets for treatment of allergic and inflammatory diseases. The specific roles of PI3K isoforms in TLR-mediated activation of lymphocytes have not been defined. In this study we assess the role of p110 delta PI3K in TLR4, TLR9, or TLR4+TLR9-mediated B cell responses. Utilizing both p110 delta-mutant mice and p110 delta-specific inhibitor IC87114, we find that signaling via p110 delta is required for optimal B cell proliferation, but is not required for TLR-mediated B cell differentiation into plasma cells or Ig isotype switch. However PI3K blockade led to increased frequencies of IgG1 and IgE expressing cells, and partially reversed ability of CpG to inhibit IgG1 and IgE. Examination of B cell cytokine production revealed that p110 delta blockade markedly reduced IL-6 and IL-10 production. In contrast, p110 delta signaling was clearly not required for IL-12 production, with p110 delta-mutant B cells in fact showing enhanced IL-12 p70 production. TLR4- and TLR9-ligands act in synergy to drive IL-6 and IL-10 production, but not IL-12, and this additive effect is independent of p110 delta signaling. Together, these results indicate that PI3K delta functions in influencing the type of B cell cytokine production and differentiation response induced by TLR-ligands.

Requirement for Phosphoinositide 3-Kinase p110δ Signaling in B Cell Antigen Receptor-Mediated Antigen Presentation

The BCR serves to both signal cellular activation and enhance uptake and presentation of Ags by B cells; however, the intracellular signaling mechanisms linking the BCR to Ag presentation functions have been controversial. PI3Ks are critical signaling enzymes controlling many cellular processes, with the p110δ isoform playing a critical role in BCR signaling. In this study, we used pharmacological and genetic approaches to evaluate the role of p110δ signaling in Ag presentation by primary B lymphocytes. It was found that activation of allogeneic T cells is significantly reduced when B cells are pretreated with global PI3K inhibitors, but was intact when p110δ signaling was specifically inactivated. In contrast, inactivation of p110δ significantly impaired the ability of B cells to activate T cells in a BCR-mediated Ag uptake and presentation model. Prestimulation of p110δ-inactivated B cells with anti-CD40 or LPS could not rescue their BCR-mediated Ag presentation ability to normal levels. p110δ signaling was required for efficient presentation of either anti-Ig or protein Ag via a lysozyme-specific BCR. p110δ-inactivated B cells were able to internalize Ag normally, and no defects in association of Ag with lysosome-associated membrane protein 1+ late endosomes were observed; however, these cells were less effective in forming polarized conjugates with Ag-specific T cells. Our data demonstrate a role for p110δ signaling in B cell Ag presentation function, implicating 3-phosphoinositides and their targets in the latter stages of this process.