Autoantibody-associated kappa light chain variable region gene expressed in chronic lymphocytic leukemia with little or no somatic mutation. Implications for etiology and immunotherapy.

Recently the minor B cell subpopulation that expresses the CD5 (Leu-1) antigen has been implicated as a source of IgM autoantibodies. Chronic lymphocytic leukemia (CLL), the most common leukemia in humans, represents a malignancy of small B lymphocytes that also express the CD5 antigen. However, little is known concerning the antibody variable region genes (V genes) that are used by these malignant CD5 B cells. We have found that a relatively high frequency of CLL patients have leukemic B cells with surface immunoglobulin (sIg) recognized by 17.109, a murine mAb specific for a kappa light chain associated crossreactive idiotype (CRI) associated with rheumatoid factor and other IgM autoantibodies. Flow cytometric analyses revealed that the relative expression of the 17.109-CRI by circulating leukemic B cells was directly proportional to the levels of sIg kappa light chain, indicating that there exists stable idiotype expression in the leukemic population. To examine this at the molecular level, the nucleic acid sequences encoding the Ig kappa light chains of two unrelated patients with CLL bearing sIg with the 17.109-CRI were determined. Analyses of multiple independent kappa light chain cDNA clones did not reveal any evidence for sequence heterogeneity in the CLL cell population. Furthermore, the nucleic acid sequences expressed by the leukemic cells of these two patients were identical or very homologous to a germline V kappa gene isolated from placental DNA, designated Humkv 325, or "V kappa RF" because of its association with IgM autoantibodies. This study suggests; (a) that the malignant CD5+ B lymphocytes in CLL use the same V kappa gene that has been highly associated with IgM autoantibodies and (b) that the expression of V genes is stable in CLL, in contrast to other B cell malignancies examined to date. We propose that many CLL cases represent malignancies of autoreactive CD5 B cells that use a restricted set of conserved V genes. This property may render CLL particularly amenable to immunotherapy with antiidiotypic antibodies

Cross-Desensitization of Chemoattractant Receptors Occurs at Multiple Levels: Evidence for a Role for Inhibition of Phospholipase C Activity

To define the molecular mechanisms of cross-regulation among chemoattractant receptors, we stably coexpressed, in a rat basophilic leukemia (RBL-2H3) cell line, epitope-tagged receptors for the chemoattractants formylmethionylleucylphenylalanine (fMLP), a peptide of the fifth component of the complement system (C5a), and interleukin-8 (IL-8). All the expressed receptors underwent homologous phosphorylation and desensitization upon agonist stimulation. When co-expressed, epitope-tagged C5a receptor (ET-C5aR) and epitope-tagged IL-8 receptor (ET-IL-8RA) were cross-phosphorylated by activation of the other. Activation of epitope- tagged fMLP receptor (ET-FR) also cross-phosphorylated ET-C5aR and ET-IL- 8RA, but ET-FR was totally resistant to cross-phosphorylation. Similarly, C5a and IL-8 stimulation of [35S]guanosine 5\u27-3-P-(thio) triphosphate (GTPγS) binding and Ca2+ mobilization were cross-desensitized by each other and by fMLP. Stimulation of [35S]GTPγS binding by fMLP was also not cross- desensitized by C5a or IL-8, however, Ca2+ mobilization was, suggesting a site of inhibition distal to G protein activation. Consistent with this desensitization of Ca2+ mobilization, inositol 1,4,5-trisphosphate release in RBL-2H3 cells expressing both ET-C5aR and ET-FR revealed that fMLP and C5a cross-desensitized each other\u27s ability to stimulate phosphoinositide hydrolysis. Taken together, these results indicate that receptor cross- phosphorylation correlates directly with desensitization at the level of G protein activation. The ET-FR was resistant to this process. Of note, cross- desensitization of ET-FR at the level of phosphoinositide hydrolysis and Ca2+ mobilization was demonstrated in the absence of receptor phosphorylation. This suggests a new form of chemoattractant cross-regulation at a site distal to receptor/G protein coupling, involving the activity of phospholipase C

The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores

P2Y2 receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemoattractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide. UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2 receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126, supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca2+-ATPases in mammalian cells, the effect of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils

Autoantibody-associated kappa light chain variable region gene expressed in chronic lymphocytic leukemia with little or no somatic mutation. Implications for etiology and immunotherapy.

Recently the minor B cell subpopulation that expresses the CD5 (Leu-1) antigen has been implicated as a source of IgM autoantibodies. Chronic lymphocytic leukemia (CLL), the most common leukemia in humans, represents a malignancy of small B lymphocytes that also express the CD5 antigen. However, little is known concerning the antibody variable region genes (V genes) that are used by these malignant CD5 B cells. We have found that a relatively high frequency of CLL patients have leukemic B cells with surface immunoglobulin (sIg) recognized by 17.109, a murine mAb specific for a kappa light chain associated crossreactive idiotype (CRI) associated with rheumatoid factor and other IgM autoantibodies. Flow cytometric analyses revealed that the relative expression of the 17.109-CRI by circulating leukemic B cells was directly proportional to the levels of sIg kappa light chain, indicating that there exists stable idiotype expression in the leukemic population. To examine this at the molecular level, the nucleic acid sequences encoding the Ig kappa light chains of two unrelated patients with CLL bearing sIg with the 17.109-CRI were determined. Analyses of multiple independent kappa light chain cDNA clones did not reveal any evidence for sequence heterogeneity in the CLL cell population. Furthermore, the nucleic acid sequences expressed by the leukemic cells of these two patients were identical or very homologous to a germline V kappa gene isolated from placental DNA, designated Humkv 325, or "V kappa RF" because of its association with IgM autoantibodies. This study suggests; (a) that the malignant CD5+ B lymphocytes in CLL use the same V kappa gene that has been highly associated with IgM autoantibodies and (b) that the expression of V genes is stable in CLL, in contrast to other B cell malignancies examined to date. We propose that many CLL cases represent malignancies of autoreactive CD5 B cells that use a restricted set of conserved V genes. This property may render CLL particularly amenable to immunotherapy with antiidiotypic antibodies

Myocardial overexpression of GRK3 in transgenic mice: evidence for in vivo selectivity of GRKs.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase 3 (GRK3) to explore the in vivo role of this GRK in cardiac function. GRK3 is expressed in the heart along with the beta-adrenergic receptor kinase (beta-ARK1) and GRK5. We have previously demonstrated that myocardial-targeted overexpression in transgenic mice of beta-ARK1 (Koch, W.J., H. A. Rockman, P. Samama, R. A. Hamilton, R. A. Bond, C. A. Milano, and R. J. Lefkowitz. Science 268: 1350-1353, 1995) or GRK5 (Rockman, H.A., D.-J. Choi, N. U. Rahman, S. A. Akhter, R. J. Lefkowitz, and W. J. Koch. Proc. Natl. Acad. Sci. USA 93: 9954-9959, 1996) results in significant attenuation of beta-adrenergic signaling and in vivo cardiac function and selective desensitization of angiotensin (ANG) II-mediated cardiac responses. Surprisingly, myocardial overexpression of GRK3 resulted in normal biochemical signaling through beta-adrenergic receptors (beta-ARs), and in vivo hemodynamic function in response to a beta-AR agonist was indistinguishable from that in nontransgenic controls. Furthermore, in vivo signaling and functional responses to ANG II were unaltered. However, myocardial thrombin signaling, as assessed by p42/p44 mitogen-activated protein (MAP) kinase activation, was significantly attenuated in GRK3 transgenic mouse hearts, indicating a distinct in vivo substrate specificity for GRK3