ABSTRACT
The accumulation of G1 cell cycle-related proteins by resting or cycling B cells stimulated with B cell antigen receptor (BCR)- and T helper (Th) cell-derived signals is documented. Resting B cells constitutively express cyclin dependent kinase (cdk)4, cdk2 and the cyclin dependent kinase inhibitor (CKI), p27. The initiation of optimal proliferation with F(ab')2 anti-mu plus paraformaldehyde-fixed CD40 ligand-baculovirus-infected Sf9 cells (CD40L/Sf9 cells) increases accumulation of both cdk4 and cdk2 while decreasing p27 levels. B cells express cyclin D2 early during cycle progression, while cyclin D3 and E are not expressed until 18 h poststimulation and cyclin A by 24 h poststimulation. Cycling B cells express heightened levels of all these cyclins and cdks. Although neither BCR- nor CD40-mediated signals appreciably alter cycling B cell accumulation of cyclins D2, cdk4 and cdk2, the absence of BCR-derived signals results in a decreased accumulation of cyclins D3 and E. Finally, CD40-mediated signals induce resting B cells to accumulate the CKI, p21, while cycling B cells require both BCR- and CD40-mediated signals to maintain increased expression of p21. Thus, a Th cell-derived signal may impact upon both resting and cycling B cell cycle progression, at least in part, by regulating the accumulation of p21. The functional consequences of p21 accumulation as cells enter and move through the cell cycle are discussed.
Subject(s)
B-Lymphocytes/cytology , CD40 Antigens/physiology , Cell Cycle Proteins/biosynthesis , Cell Cycle/physiology , Cyclins/biosynthesis , Lymphocyte Activation/physiology , Muscle Proteins , Tumor Suppressor Proteins , Animals , B-Lymphocytes/metabolism , Cell Cycle Proteins/genetics , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclins/genetics , Female , Gene Expression Regulation , Ionomycin/pharmacology , Ionophores/pharmacology , Lymphocyte Cooperation , Mice , Mice, Inbred DBA , Microfilament Proteins/biosynthesis , Microfilament Proteins/genetics , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Proteins , Receptors, Antigen, B-Cell/immunology , T-Lymphocytes, Helper-Inducer/immunology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Because isolation of sufficient numbers of cycling, germinal center B cells from mice for biochemical characterization of BCR-derived signals can be problematic, we have designed an experimental approach for generating large numbers of cycling B cells for further study. In the experiments reported here, small, resting B cells were polyclonally stimulated with lipopolysaccharide (LPS), and cycling B cells isolated as two bands on three-step Percoll gradients. Cycling B cells isolated at Days 2, 4, or 6 of preactivation showed an increased expression of Fas receptor and peanut agglutinin binding, with a concomitant decrease in sIgD positivity. These cells phenotypically resembled extrafollicular or early germinal center B cells. These cycling B cells were used to study the functional consequences of differential signaling through the BCR. Strong cross-linking of BCR, by restimulation of cycling normal B cells with either immobilized or soluble F(ab')2 anti-mu and cycling hen egg lysozyme (HEL) transgenic B cells with either soluble or immobilized HEL, extended cellular proliferation by 2-3 d. In contrast, cycling B cells either restimulated with soluble, whole anti-mu (to mimic binding of soluble immune complexes) or cultured in the absence of restimulation (to mimic cycling B cells not competitive for antigen) resulted in the rapid exit of the cells from cycle. This system will enable the molecular and biochemical characterization of signal delivery to cycling B cells.
Subject(s)
B-Lymphocyte Subsets/cytology , Animals , B-Lymphocyte Subsets/immunology , Cell Cycle , Cells, Cultured , Female , Flow Cytometry , Immunophenotyping , Lipopolysaccharides/pharmacology , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Spleen/cytologyABSTRACT
Calcitonin gene-related peptide, (CGRP), a vasoactive neuropeptide, is found throughout the peripheral nervous system, and CGRP receptors are present on mature lymphocytes. The current studies describe a CGRP receptor on isolated mouse bone marrow cells. The affinity, distribution and specificity of CGRP receptors were analyzed using radioligand binding assays. [125I]CGRP binding in mouse bone marrow cells was dependent on cell concentration and was stable from 5 to 60 min at room temperature. The average Kd is 3.29 +/- 1.24 nM and the average receptor density is 2796 +/- 365 sites/cell. Competition binding analysis found rat alpha and beta CGRP to be the most inhibitory, (Ki values 0.899 and 0.711 nM, respectively), followed by human alpha CGRP and the antagonist human CGRP8-37. The neuropeptides human and salmon calcitonin did not inhibit [125I]CGRP binding to bone marrow cells. The presence of CGRP receptors on mouse bone marrow cells provides further evidence for a direct role for CGRP in modulating the function and differentiation of cellular components of the immune and inflammatory systems.
