Subbarao Bondada

CD5-Mediated Negative Regulation of Antigen Receptor-Induced Growth Signals in B-1 B Cells

A subset of B lymphocytes present primarily in the peritoneal and pleural cavities is defined by the expression of CD5 and is elevated in autoimmune diseases. Upon signaling through membrane immunoglobulin M (mIgM), splenic B lymphocytes (B-2) proliferate, whereas peritoneal B cells (B-1) undergo apoptosis. However, in CD5-deficient mice, B-1 cells responded to mIgM crosslinking by developing a resistance to apoptosis and entering the cell cycle. In wild-type B-1 cells, prevention of association between CD5 and mIgM rescued their growth response to mIgM crosslinking. Thus the B cell receptor-mediated signaling is negatively regulated by CD5 in normal B-1 cells.

Rapamycin, a potent immunosuppressive drug, causes programmed cell death in B lymphoma cells

Rapamycin, a potent immunosuppressive drug that prevents rejection of organ transplants in many animals, caused profound growth inhibition in an immature B cell lymphoma, BKS-2, at very low concentrations (2 ng/ml). Similar growth inhibition was also observed in a series of B cell lymphomas (i.e., L1.2, NFS.1.1, and WEHI-279) as well as in thymoma cells. The cell death induced by rapamycin in BKS-2 lymphoma was found to be via programmed cell death, or apoptosis. In contrast to rapamycin, neither FK506 nor CsA affected the normal growth of these cells. FK506, but not CsA antagonized the effect of rapamycin and rescued the BKS-2 cells from undergoing apoptosis. Further, suboptimal concentrations of antiIgM antibodies and rapamycin acted synergistically in causing the growth inhibition of BKS-2 cells and this inhibitory effect was also completely reversed by FK506. Thus, rapamycin appeared to inhibit lymphoma growth by binding to FK506 binding protein. These results indicate that rapamycin should be evaluated as an effective immunosuppressive therapeutic agent to prevent the incidence of lymphoma after transplantations.

The unresponsiveness of aged mice to polysaccharide antigens is a result of a defect in macrophage function

A reduction in macrophage (MΦ) function with aging makes mice less responsive to bacterial capsular polysaccharides, such as those present in the pneumococcal polysaccharide vaccine, a model of thymus independent (TI) antigen (Ag). Using trinitrophenol (TNP)‐lipopolysaccharide (LPS) and TNP‐Ficoll, two other well‐studied TI Ag, we studied the mechanistic basis of reduced MΦ function in the aged. We show that aged mice are profoundly hyporesponsive to these TI Ag. As a result of a requirement for MΦ, highly purified B cells from young‐adult mice do not respond to TI Ag. When purified, young B cells were immunized with TNP‐Ficoll, the antibody production from those cultures reconstituted with MΦ from aged mice was significantly lower than that seen with young MΦ. Consequently, this unresponsiveness can be overcome by a mixture of interleukin (IL)‐1β and IL‐6. Upon stimulation with LPS, in comparison with young MΦ, aged MΦ secreted reduced amounts of IL‐6, tumor necrosis factor α, IL‐1β, and IL‐12, cytokines necessary for B cells to respond to TI Ag. LPS also induced aged MΦ to produce an excess of IL‐10. Neutralization of IL‐10 enhanced the production of proinflamatory cytokines by MΦ upon LPS stimulation and also induced Ab production by aged splenocytes. Thus, the inability of aged MΦ to help the B cell response appears to be caused by an excess of IL‐10. As aged MΦ have a reduced number of cells expressing Toll‐like receptor 4 and CD14, the imbalance in cytokine production might be partly a result of fewer cells expressing key components of the LPS receptor complex.

Molecular basis of age-associated cytokine dysregulation in LPS-stimulated macrophages

Aged humans and rodents are susceptible to infection with Streptococcus pneumoniae bacteria as a result of an inability to make antibodies to capsular polysaccharides. This is partly a result of decreased production of proinflammatory cytokines and increased production of interleukin (IL)‐10 by macrophages (MΦ) from aged mice. To understand the molecular basis of cytokine dysregulation in aged mouse MΦ, a microarray analysis was performed on RNA from resting and lipopolysaccharide (LPS)‐stimulated MΦ from aged and control mice using the Affymetrix Mouse Genome 430 2.0 gene chip. Two‐way ANOVA analysis demonstrated that at an overall P < 0.01 level, 853 genes were regulated by LPS (169 in only the young, 184 in only the aged, and 500 in both). Expression analysis of systematic explorer revealed that immune response (proinflammatory chemokines, cytokines, and their receptors) and signal transduction genes were specifically reduced in aged mouse MΦ. Accordingly, expression of Il1 and Il6 was reduced, and Il10 was increased, confirming our previous results. There was also decreased expression of interferon‐γ. Genes in the Toll‐like receptor‐signaling pathway leading to nuclear factor‐κB activation were also down‐regulated but IL‐1 receptor‐associated kinase 3, a negative regulator of this pathway, was increased in aged mice. An increase in expression of the gene for p38 mitogen‐activated protein kinase (MAPK) was observed with a corresponding increase in protein expression and enzyme activity confirmed by Western blotting. Low doses of a p38 MAPK inhibitor (SB203580) enhanced proinflammatory cytokine production by MΦ and reduced IL‐10 levels, indicating that increased p38 MAPK activity has a role in cytokine dysregulation in the aged mouse MΦ.

Negative regulation of antigen receptor‐mediated signaling by constitutive asociation of CD5 with the SHP‐1 protein tyrosine phosphatase in B‐1 B cells

CD5, a membrane‐associated glycoprotein, has been shown to negatively regulate antigen receptor‐mediated growth responses in peritoneal B lymphocytes, thymocytes and mature T cells. The CD5‐expressing peritoneal B cells (B‐1) that are normally unresponsive to B cell receptor (BCR)‐mediated growth signals mount a proliferative response to BCR cross‐linking if the CD5 gene is deleted or if the CD5 molecule is sequestered away from the BCR. SHP‐1, a cytosolic protein tyrosine phosphatase, has also been implicated in the negative regulation of antigen receptor‐mediated signaling. The present study shows that SHP‐1 is constitutively associated with the BCR in B‐1 cells. This association is mediated in part by CD5, as it is reduced substantially after antigen receptor ligation in CD5– / – B‐1 cells, and upon sequestration of CD5 from the antigen receptor complexes in wild‐type B‐1 cells. Prior cross‐linking of CD5 also restores a normal calcium mobilization response as well as NF‐κB activation in B‐1 cells. These data support a model whereby CD5 negatively regulates antigen receptor‐mediated growth signals by recruiting SHP‐1 into the BCR complex in B‐1 cells.

Cutting Edge: Constitutive B Cell Receptor Signaling Is Critical for Basal Growth of B Lymphoma

B lymphomas account for the majority of the lymphoma cases. BCR expression appears to be important for B lymphoma because most oncogenes are translocated to nonrearranged Ig loci and because all of the variants that arise in anti-idiotypic Ab-treated lymphoma patients remain BCR positive. Based on this and the fact that BCR is required for mature B cell survival, we tested the requirement for continued expression of BCR for the growth and survival of B lymphoma cells. Using Igα or Igβ-specific small interfering RNA (siRNA) to inhibit BCR expression, we demonstrate for the first time that constitutive signaling by BCR is critical for survival and proliferation of both murine and human B lymphoma cells. The BCR signals in lymphoma appear to be mediated by Syk, as it is constitutively active in a variety of B lymphoma cells. Blocking Syk activity by selective inhibitors suppresses growth of several murine and human B lymphomas.

Defective macrophage function in neonates and its impact on unresponsiveness of neonates to polysaccharide antigens

Neonates do not respond to thymus‐independent (TI) antigens (Ag), making them vulnerable to infection with encapsulated bacteria. The antibody (Ab) response of adult and neonatal B cells to TI Ag requires certain cytokines, which are provided by T cells or macrophages (MΦ). Lipopolysaccharide (LPS) failed to induce neonatal MΦ to produce interleukin (IL)‐1β and tumor necrosis factor α (TNF‐α) mRNA and to secrete IL‐1β, IL‐12, and TNF‐α. However, LPS induced neonates to secrete some IL‐6 and three‐ to fivefold more IL‐10 than adults. Accordingly, adding adult but not neonatal MΦ could restore the response of purified adult B cells to trinitrophenol (TNP)–LPS, a TI Ag. Increased IL‐10 is causally related to decreased IL‐1β and IL‐6 production, as IL‐10−/− neonatal MΦ responded to LPS by secreting more IL‐1β and IL‐6 than wild‐type (WT) neonatal MΦ. When cultures were supplemented with a neutralizing Ab to IL‐10, WT neonatal MΦ secreted increased amounts of IL‐6 and allowed neonatal MΦ to promote adult B cells to mount an Ab response against TNP–LPS. Thus, neonates do not respond to TI Ag as a result of the inability of neonatal MΦ to secrete cytokines, such as IL‐1β and IL‐6, probably as a result of an excess production of IL‐10. This dysregulated cytokine secretion by neonatal MΦ may be a result of a reduction in expression of Toll‐like receptor‐2 (TLR‐2) and TLR‐4 and CD14.

Spleen Tyrosine Kinase (Syk), a Novel Target of Curcumin, Is Required for B Lymphoma Growth

Curcumin (diferuloylmethane), a component of dietary spice turmeric (Curcuma longa), has been shown in recent studies to have therapeutic potential in the treatment of cancer, diabetes, arthritis, and osteoporosis. We investigated the ability of curcumin to modulate the growth of B lymphomas. Curcumin inhibited the growth of both murine and human B lymphoma in vitro and murine B lymphoma in vivo. We also demonstrate that curcumin-mediated growth inhibition of B lymphoma is through inhibition of the survival kinase Akt and its key target Bad. However, in vitro kinase assays show that Akt is not a direct target of curcumin. We identified a novel target for curcumin in B lymphoma viz spleen tyrosine kinase (Syk). Syk is constitutively activated in primary tumors and B lymphoma cell lines and curcumin down-modulates Syk activity accompanied by down-regulation of Akt activation. Moreover, we show that overexpression of Akt, a target of Syk, or Bcl-xL, a target of Akt can overcome curcumin-induced apoptosis of B lymphoma cells. These observations suggest a novel growth promoting role for Syk in lymphoma cells.

Hippocampal neurons of mice deficient in DNA-dependent protein kinase exhibit increased vulnerability to DNA damage, oxidative stress and excitotoxicity

DNA damage has been documented in neurodegenerative conditions ranging from Alzheimers disease to stroke. DNA-dependent protein kinase (DNA-PK) is involved in V(D)J recombination and DNA double strand break repair, and may play a role in cell death induced by DNA damage. We now report that cultured hippocampal neurons from severe combined immunodeficient (scid) mice which lack DNA-PK activity are hypersensitive to apoptosis induced by exposure to topoisomerase inhibitors, amyloid beta peptide (A beta) and glutamate. A similar increased vulnerability of hippocampal CA1 and CA3 neurons was observed in adult scid mice after kainate-induced seizures. Our results suggest that DNA-PK activity is important for neuron survival under conditions that may occur in neurological disorders.

Accessory cell defect in unresponsiveness of neonates and aged to polysaccharide vaccines

T independent antigens elicit antibody responses in the absence of carrier specific T helper cells but require signals from accessory cells (macrophages and dendritic cells) or specific cytokines. They are further subdivided into TI-1 and TI-2 categories based on the ability of TI-1 but not TI-2 antigens to elicit immune responses from neonates. Most bacterial polysaccharides including the pneumococcal polysaccharide vaccines belong to the TI-2 class. It is hypothesized that defects in accessory cell function play a critical role in the failure of neonates to respond to such TI-2 antigens. Immune responses to these TI-2 stimuli are also reduced in the aged, also due to a quantitative deficiency in accessory cells. Agents that can stimulate accessory cell function may provide an alternative strategy to improve the immunogenicity of the polysaccharide vaccines in the neonates and the aged.