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Originally published online as doi:10.1189/jlb.0306229 on November 16, 2006

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(Journal of Leukocyte Biology. 2007;81:802-808.)
© 2007 by Society for Leukocyte Biology

Calcitonin gene-related peptide inhibits early B cell development in vivo

Jerome J. Schlomer, Benjamin B. Storey, Radu-Tudor Ciornei and Joseph P. McGillis1

Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA

1 Correspondence: MS 401, Department of Micro-Immunol, University of Kentucky College of Medicine, 800 Rose St., Lexington, KY 40536, USA. E-mail: jpmcgi01{at}uky.edu


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ABSTRACT
 
Recent in vitro studies suggest that calcitonin gene-related peptide (CGRP) inhibits early B cell differentiation; however, there is no evidence in the intact animal for a role for CGRP in B cell development. Here, we show that in vivo treatment of mice with CGRP reduces the number of IL-7 responsive B cell progenitors in bone marrow. A single CGRP treatment reduces IL-7-responsive B cell progenitors by up to 40% for up to 72 h. The reduction is dose-dependent and can be blocked by a CGRP receptor antagonist, CGRP8–37. CGRP in serum following injection is highly elevated at 30 min but returns to basal levels by 4 h, suggesting that a single injection of CGRP has long-lasting effects on B cell development. This report provides the first direct in vivo evidence that CGRP, a neuropeptide with multiple effects on mature lymphocytes, also plays a regulatory role in early B cell development in the bone marrow.

Key Words: neuropeptides • lymphopoiesis • hematopoiesis • neuroimmunology


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INTRODUCTION
 
Calcitonin gene-related peptide (CGRP) is a 37-amino acid sensory neuropeptide, which is released from nociceptive neurons in local microenvironments [1 ]. It is widely distributed in nociceptive nerve endings throughout the body and is frequently colocalized with substance P (SP) [2 , 3 ]. It is derived from a precursor gene whose heterogeneous nuclear RNA is spliced differentially to give rise to a calcitonin precursor mRNA in endocrine tissue or a CGRP precursor mRNA in neural tissue [4 ]. Its release at local sites of inflammation is stimulated by mechanical and chemical stimuli and by inflammatory mediators [5 6 7 ]. CGRP, along with SP, induces many of the phenomena associated with neurogenic inflammation—vasodilation, fluid extravasation, and regulatory effects on inflammatory cells [8 ]. Receptors for CGRP are expressed on lymphocyte and monocyte lineages, and CGRP influences a number of mature T cell, B cell, and macrophage functions in vitro [9 10 11 12 13 14 ]. These include proliferative responses, antigen presentation, surface protein expression, and cytokine production [15 16 17 18 19 ]. Although the effects of CGRP on mature cell functions are fairly well-documented, recent in vitro studies suggest that CGRP also influences early B cell development [14 , 20 21 22 ].

B cell differentiation from hematopoietic stem cells involves the ordered progression through several distinct stages [23 ]. Several key events occur at specific stages, including light- and heavy-chain Ig gene rearrangement, and must be completed before the developing B cell can progress to subsequent stages. Following successful rearrangement of the Ig heavy-chain gene in the mouse, IL-7 stimulates several rounds of cell division [24 ]. Rearrangement of the Ig light-chain gene follows this cell division, and in the mouse, it is estimated that 95% of the developing B cells which have rearranged their heavy chains successfully rearrange their light-chain genes. Cells that have successfully rearranged heavy and light chains can progress to the immature B cell stage (IgM+/IgD–), characterized by expression of the BCR. If the BCR, on an immature B cell is engaged by its cognate antigen (typically a self-antigen) the cell undergoes clonal deletion.

In vitro studies with cell lines and cells from bone marrow suggest that CGRP has inhibitory influences on early B cell development. First, CGRP inhibits IL-1- or LPS-induced expression of the BCR on the 70z/3 pre-B cell line [20 ]. The 70Z/3 pre-B cell line has been used to study cellular events in the transition from a BCR– pre-B cell phenotype to a BCR+ immature phenotype [25 ]. More recent in vitro studies using normal bone marrow found that CGRP inhibits the proliferative response of B cell progenitors to IL-7 [21 ]. It does this by acting directly on B cell progenitors as well as by inducing cytokines in the stroma (IL-6 and TNF-{alpha}) which inhibit B cell progenitor-proliferative responses to IL-7 [21 , 22 ]. In spite of growing in vitro evidence for a role for CGRP in early B cell development, there is no evidence that CGRP has any effect on B cell development in the intact animal. In the current studies, the effect of in vivo CGRP treatment on ex vivo responses of late pro-B cells in bone marrow to IL-7 was evaluated. It was observed that i.v. treatment of mice with CGRP causes a significant and transient decrease in the number of pre-B CFUs in bone marrow.


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MATERIALS AND METHODS
 
Materials
Male, 6- to 8-week-old BALB/c mice were obtained from National Cancer Institute (Bethesda, MD, USA) or Charles River Laboratories (Wilmington, MA, USA). CGRP and other neuropeptides were obtained from Bachem, Inc. (Torrance, CA, USA). Rat {alpha}CGRP was used in these studies. In previous studies, we observed no difference in lymphocyte responses to {alpha}- and ßCGRP or in mouse lymphocyte CGRP receptor affinity for the {alpha} and ß forms [10 ]. Mouse and rat {alpha}- and ßCGRPs are identical, and the {alpha} and ß forms differ by 1 amino acid (Glu vs. Lys at Position 35). Antibodies for FACS analysis were obtained from PharMingen (San Diego, CA, USA), and anti-CGRP for RIAs was obtained from Peninsula Laboratories (Belmont, CA, USA). IL-7 was purchased from R&D Systems (Minneapolis, MN, USA).

CGRP treatment
All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee. Prior to i.v. injections, mice were anesthetized by i.p. injection of ketamine/xylazine/acepromazine. Neuropeptides were diluted in PBS to the desired concentration and administered i.v. by injection of 100 ul into the retro-orbital sinus. After the treatment period, bone marrow was collected from femurs and was used for analysis of pre-B CFUs or for FACS analysis. For studies of CGRP levels in serum, trunk blood was collected, and serum was stored at –20ºC until analysis by RIA.

Pre-B CFU assays
Pre-B CFU assays, based on the procedure described by Lee et al. [26 ], were used to assess IL-7 responses in bone marrow. Briefly, bone marrow was collected from femurs, and RBCs were depleted by lysis in NH4Cl. RBC-depleted bone marrow cells (100,000) were plated in soft agar in 3 cm culture dishes in the presence or absence of IL-7 at 1 ng/ml. The dishes were incubated at 37ºC for 6 days, and CFUs were counted on an inverted microscope. Each CFU consisted of a compact cluster of at least 40 cells. Plates that did not receive IL-7 had zero to three colonies, and plates that received IL-7 from PBS-treated animals had from 80 to 130 CFUs. Approximately 95% of the cells that successfully rearrange their Ig heavy chain also successfully rearrange an Ig light chain after responding to IL-7. Thus, the number of IL-7-responding cells provides one relative measure of the rate of new B cell production. Where data are presented from multiple independent experiments, the results are normalized with the IL-7 response in PBS-treated animals being set at 100%.

Flow cytometry
Analysis of early B cell progenitors by four-color flow cytometry was done as described by Hardy et al. [27 ] with modifications in fluorochromes to accommodate the lasers available on our FACS instruments. Briefly, duplicate tubes of RBC-depleted bone marrow cells from each mouse were incubated with anti-B220-Cy-Chrome, anti-CD43-FITC, biotinylated anti-CD24, and anti-BP-1-PE for 1 h on ice, followed by streptavidin-allophycocyanin for 30 min on ice. Analysis was done in the University of Kentucky Flow Cytometry Core Facility (Lexington) on a BD FACSCaliber using CellQuest software (Becton Dickinson, San Jose, CA, USA) for analysis. At least 100,000 cells were analyzed for each sample. Cells in Fractions A–C are B220+/CD43+ (cells in Fraction A are CD24–/BP-1–, cells in Fraction B are CD24+/BP-1–, and cells in Fraction C are CD24+/BP-1+).

CGRP RIA
CGRP levels in serum were measured using a RIA for CGRP in serum, which has been described previously and validated in our laboratory for analysis of CGRP in human serum [28 ]. Briefly, serum from treated mice was diluted in RIA buffer to dilutions that gave values on the linear portion of the standard curve. The serum was incubated for 24 h at 4ºC, and anti-CGRP and HPLC-purified 125I-CGRP were prepared as described previously [29 ]. Bound 125I-CGRP was precipitated by incubation with Pansorbin cells followed by centrifugation. CGRP concentrations in serum dilutions were estimated by extrapolation from a standard curve of murine {alpha}CGRP. The RIA recognizes {alpha}- and ßCGRP equivalently.

Data analysis
Statistical analysis was done by two-tailed Student’s t-test between CGRP and control-treated groups. Statistical significance was accepted at P < 0.05.


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RESULTS
 
Based on the ability of CGRP to inhibit proliferation of B cell progenitors in vitro [21 ], the first goal was to determine whether in vivo treatment of mice with CGRP affects developing B cells in bone marrow. For these studies, mice were treated i.v. with CGRP, and B cell development was assessed with a pre-B cell CFU assay [26 ]. In freshly isolated bone marrow, this assay provides an assessment of the number of cells that will respond to IL-7 by proliferating. Cells that respond to IL-7 by proliferating are in the late pro-B cell stage and have rearranged their heavy-chain genes successfully [30 ]. As most of the proliferating cells will rearrange their light-chain genes successfully, it provides a relative estimate of the number of cells that will progress to the immature B cell stage. To determine the kinetics of inhibition of pre-B CFUs by CGRP, a single injection of CGRP was given i.v. at a dose of 100 nmol/kg, and pre-B CFUs were measured at times from 1 h to 96 h. As shown in Figure 1 A, pre-B CFU levels in bone marrow were decreased up to 40% at time-points from 6 h to 72 h following CGRP treatment. Pre-B CFU levels returned to baseline by 96 h. Figure 1A shows the pooled data from several individual experiments. The number of pre-B CFUs in individual experiments was normalized against the IL-7 response in PBS-treated animals, and the IL-7 response was set at 100%. The number of pre-B CFUs in individual experiments typically ranged from 80 to 120 pre-B CFUs/105 bone marrow cells.


Figure 1
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  		Figure 1. CGRP inhibits IL-7 responses in vivo. (A) Kinetic analysis of in vivo inhibition of IL-7 pre-B CFU responses by CGRP. PBS or CGRP in PBS (100 µl; 100 nmol/kg) was injected i.v. Bone marrow was collected at 1–96 h, and IL-7 pre-B CFUs were analyzed by pre-B CFU assay. Data from individual experiments were normalized versus the response to IL-7 in PBS-treated animals at the same time-point, and the PBS response was set at 100%. Each individual data point represents the mean ± SEM from three to six independent experiments. In individual experiments, each treatment group contained eight animals. IL-7 responses were significantly suppressed (P<0.05) at times ranging from 6 h to 72 h. (B) Concentration dependence of CGRP inhibition of IL-7 responses. PBS or CGRP at 5–200 nmol/kg body weight were injected i.v. Bone marrow was collected at 24 h, and IL-7 responses were analyzed in the pre-B CFU assay. Each treatment group included eight animals, and each data point is the mean CFUs/105 bone marrow cells ± SEM. CGRP induced a concentration-dependent decrease in the number of IL-7-responsive cells.

The inhibitory effect of CGRP on the number of IL-7-responsive cells in bone marrow is also concentration-dependent. A representative dose response experiment is shown in Figure 1B . Mice were treated with CGRP at doses ranging from 5 to 200 nmol/kg, and pre-B CFUs were measured 24 h later [24 h was selected, as the maximal inhibitory effect is observed by 24 h (Fig. 1A) ]. The effect of CGRP given i.v. was found to be dose-dependent, and optimal inhibition was observed at doses above 50 nmol/kg.

To determine whether the in vivo effect of CGRP on early B cell progenitors is specific, mice were treated with calcitonin as a nonspecific peptide control or with CGRP and the CGRP antagonist CGRP8–37 [31 ]. Calcitonin, derived from the same gene as CGRP, is of similar size and charge, has little sequence homology, and does not cross-react with lymphocyte CGRP receptors [9 , 10 , 32 ]. CGRP8–37 is a truncated form of CGRP lacking the first 7 amino acids that acts as an antagonist at CGRP receptors. The peptides were administered i.v., and pre-B CFUs were measured after 24 h. As shown in Figure 2 , calcitonin had no effect on pre-B CFUs. Likewise, CGRP8–37 by itself did not inhibit pre-B CFUs. However, CGRP8–37, administered with CGRP, blocked the inhibitory effect of CGRP on bone marrow pre-B CFUs. These results show that the effect of CGRP is specific and is mediated by specific CGRP receptors.


Figure 2
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  		Figure 2. Specificity of CGRP inhibition of in vivo IL-7 responses. Mice (n=8) were injected i.v. with PBS, CGRP, CGRP8–37, or calcitonin at 100 nmol/kg or with CGRP at 100 nmol/kg and CGRP8–37at 250 nmol/kg. After 24 h, bone marrow was collected, and the number of IL-7-responsive cells was measured in the pre-B CFU assay. Each bar is the mean ± SEM, normalized with the PBS treatment set at 100%. Pre-B CFUs were reduced significantly in the CGRP-treated mice relative to PBS or CGRP + CGRP8–37 (P<0.05). Pre-B CFUs in calcitonin were not significantly different than PBS controls.

In the studies shown in Table 1 , we observed that at higher doses, CGRP8–37 could actually increase the number of IL-7 pre-B CFUs in bone marrow. Three additional experiments (Fig. 2 and data not shown) included CGRP8–37 treatments at doses between 100 and 166 nmol/kg. In one of the studies, shown in Figure 2 , CGRP8–37 at 100 nmol/kg did not show an increase in pre-B CFUs. The results of the other two studies with doses of 100 nmol/kg and 166/ nmol/kg were similar to the results of Experiment I in Table 1 . The increase in pre-B CFUs following CGRP8–37 treatment, especially at higher doses, suggests that CGRP8–37 may be blocking the inhibitory effects of endogenous CGRP. The reason that higher concentrations of CGRP8–37 are required is probably because of the short-lived presence of the antagonist. Use of a continuous delivery system, such as a minipump, may actually require less CGRP8–37 to inhibit endogenous CGRP than is required for a single injection.


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  		Table 1. Increase in Bone Marrow Pre-B CFUs in CGRP8–37-Treated Mice

In vitro studies suggest that CGRP has a direct inhibitory effect on IL-7-induced proliferation of B cell progenitors. To identify the B cell progenitor populations affected by CGRP in vivo, the phenotypic distribution of early B cell precursors in bone marrow was assessed following treatment with CGRP. Using the scheme proposed by Hardy et al. [27 ], the frequency of cells in the three earliest B cell populations, A–C, was measured by flow cytometry. The C (late pro-B) population, which has rearranged the Ig heavy-chain but not the light-chain genes, contains the subpopulation of cells that respond to IL-7 by proliferating. As can be seen in Figure 3 , there is a decrease in the frequency of cells in bone marrow in the early B cell populations A–C. Figure 3A shows a representative FACS staining for the bone marrow cells from one PBS- and one CGRP-treated animal. The upper panels show B220 and CD43 staining, and the lower panels show CD24 and BP-1 staining on B220+/CD43+ cells. Cells in the A–C populations are CD24–/BP-1–, CD24+/BP-1–, or CD24+/BP-1+, respectively. The frequency of each population in total bone marrow is shown next to each quadrant. The graph in Figure 3 shows the average frequency of cells in each population for PBS- and CGRP-treated animals (n=6 mice per treatment group). The reduction in cells in the A and B fractions also suggests that CGRP has effects on cells at the A and B and earlier stages as well as the C stage, which contains IL-7-responsive cells. No changes in the total number of bone marrow cells recovered in mice treated with CGRP were observed in the study shown in Figure 3 or in the studies shown in Figures 1 and 2 .


Figure 3
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  		Figure 3. CGRP decreases the number of early B cell progenitors in bone marrow. Twenty-four hours after PBS or CGRP (100 nmol/kg) injection, bone marrow was harvested, and the percent of cells in B cell progenitor fractions A–C was assessed using four-color FACS analysis as described by Hardy et al. [27 ]. The upper panels show B220 versus CD43 staining and the location of the gate (R2) that was analyzed for CD24 versus BP-1 expression in the lower panels. In the lower panels, cells in Fraction A are B220+/CD43+/CD24–/BP-1–, cells in Fraction B are B220+/CD43+/CD24+/BP-1–, and cells in Fraction C are B220+/CD43+/CD24+/BP-1+. Fraction C contains the subpopulation of cells that proliferate in response to IL-7 (late pro-B). A shows a representative FACS analysis for the bone marrow from one PBS- and one CGRP-treated animal with the percentage in total bone marrow of the three early fractions. (B) The mean ± SEM for frequency of cells in each fraction and an n of 6 animals per treatment. The open bars show frequency in total bone marrow in PBS-treated animals, and the hatched bars show the frequency in animals treated with CGRP. All three fractions are reduced significantly in bone marrow (P<0.005; P<0.05; P<0.02, respectively).

It was interesting that the effect of a single injection of CGRP induced changes in the B cell compartment that lasted for up to 72 h. This raised the question of how long CGRP levels are elevated following i.v. injection of CGRP, specifically, whether elevated CGRP levels were sustained for the entire period during which pre-B CFUs were decreased. In that neuropeptides are generally labile and have relatively short half-lives, the expectation is that CGRP elevation in blood will be fairly transient following i.v. injection. If this were true, it would argue that a transient exposure to elevated CGRP induces relatively long-lasting effects. To differentiate between these possibilities, CGRP levels in blood were assessed by RIA [28 ] following a single i.v. injection of CGRP, which was injected i.v. at a dose of 100 nmol/kg, and blood was collected at several time-points postinjection. As seen in Figure 4 , CGRP is elevated rapidly in blood following i.v. injection but declines to basal levels by 4 h. The rapid turnover of CGRP following i.v. injections shows that a single injection of CGRP given i.v. causes relatively long-lasting changes in the B cell compartment in bone marrow, which last well beyond the elevation of exogenous CGRP.


Figure 4
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  		Figure 4. CGRP levels in serum following i.v. injection. CGRP was injected i.v., 100 nmol/kg, and blood was collected at 0.25, 0.5, 1, 1.5, 2, and 4 h. Controls were injected with PBS, and blood was collected at 0.5 and 1 h. CGRP was measured by RIA [28 ], and each treatment group included three animals. Each data point represents the mean ± SEM; i.v. injection of CGRP at 100 nmol/kg resulted in an 80-fold increase in CGRP, which returned to basal levels by 4 h.


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DISCUSSION
 
The data in this study provide the first direct evidence in the intact animal for a regulatory role for CGRP in early B cell development. Previous in vitro studies found that CGRP inhibits IL-7 responses by direct and indirect mechanisms. Experiments using purified cells in the late pro-B cell (C) fraction, which respond to IL-7, show that CGRP directly inhibits the cells that proliferate in response to IL-7 [21 ]. In addition, it was observed that CGRP induces factors in bone marrow stromal cell cultures that inhibit IL-7-proliferative responses by late pro-B cells. Two factors, TNF-{alpha} and IL-6, were identified in supernatants from CGRP-treated stromal cells that inhibited proliferative responses to IL-7 [14 , 22 ]. The primary goal of the current studies was to demonstrate that CGRP inhibits B cell development in vivo, specifically the response to IL-7.

Based on the results in the current study, the net effect of CGRP in vivo is to decrease the number of cells that proliferate in response to IL-7 in the bone marrow, along with a decrease in the overall percentage of cells at early stages of B cell development. The effect of exogenously administered CGRP is transient. The number of IL-7 pre-B CFUs in bone marrow is decreased for up to 72 h after a single injection of CGRP, and by 96 h, the number of IL-7-responsive cells returns to baseline. Two criteria were used to determine whether the effect of CGRP was mediated through a specific CGRP receptor. In one paradigm, mice were treated with CGRP and the CGRP antagonist CGRP8–37. In the other paradigm, mice were also treated with a structurally unrelated peptide of similar size and charge, calcitonin. The ability of CGRP8–37 to block the inhibitory effect of CGRP on the B cell lineage suggests that the effects are mediated by CGRP acting on a specific CGRP receptor. The lack of an effect of calcitonin on IL-7-induced proliferation by B cells in vivo further substantiates the specificity of the inhibitory effects of CGRP. In addition, the increase in pre-B CFUs seen with higher concentrations of CGRP8–37, suggesting that it is inhibiting endogenous CGRP8–37, also supports a role for CGRP in regulating the number of IL-7-responsive B cell progenitors in bone marrow.

FACS analysis of early B cell phenotypes also suggests that CGRP reduces the steady-state levels of cells in three early B cell populations. Further studies will be necessary to determine how CGRP reduces the number of early B cells. It is likely that the reduction may result from a combination of direct effects on the B cell lineage as well as by indirect effects on stromal cells in the bone marrow. One possibility is that CGRP may be diverting cells to other lineages. This possibility will require further investigation. The reduction in cells at early stages in the B lineage supports an inhibitory role for CGRP in B cell development. The in vivo effects of CGRP on developing B cells along with the previous in vitro observations support the hypothesis that CGRP plays an inhibitory role during B cell development.

The prolonged effect of a single injection of CGRP prompted an analysis of CGRP levels in the treated mice. Neuropeptides are generally labile and are cleared fairly rapidly from local microenvironments and the circulation. Thus, it would not be expected that CGRP remains elevated for the period during which it inhibited B cell development in the bone marrow. In fact, analysis of CGRP in mice treated i.v. with CGRP found that serum levels returned to baseline by 4 h postinjection. The rapid decline in CGRP levels is not surprising based on the instability of neuropeptides in general. In fact, the effects of the sensory neuropeptides on inflammation are thought to be paracrine, acting in local microenvironments rather than by acting systemically. Although this experiment does not directly address the levels of CGRP outside the bloodstream in the bone marrow, it is not likely that elevated levels in bone marrow would be sustained much beyond the peak levels in the bloodstream. Thus, the results suggest that a relatively short exposure to elevated CGRP induces sustained changes in the B cell compartment that last for several days. An argument for the physiologic relevance of the current results can be made based on the ability of the CGRP receptor antagonist CGRP8–37 to block the effects of CGRP in vivo and on previous studies, showing that CGRP receptors are expressed on cells in the bone marrow and that CGRP has related effects on these cells in vitro [9 , 14 , 20 21 22 , 33 ]. In most cases, nonphysiologic artifact responses to high concentrations of neuropeptides are by cross-reaction at receptors other than their own or by nonreceptor-mediated mechanisms. In either case, the effects cannot be inhibited by specific receptor antagonists. Thus, reversal of the inhibitory effects of CGRP in the bone marrow by CGRP8–37 argues that the effects of CGRP are mediated by CGRP receptors in the bone marrow. A physiologic role for CGRP is also supported by the increase in pre-B CFUs in animals treated with higher concentrations of CGRP8–37.

The source of endogenous CGRP in the bone marrow is presumed to be from sensory nerve endings. Several investigators have examined CGRP in the bone and have identified CGRP-containing nerve endings in regions of the bone marrow where it could influence hematopoiesis [3 , 34 35 36 37 ]. Recent reports from one laboratory suggest that cells from hematopoietic lineages could also produce CGRP [38 , 39 ]. However, we have been unable to detect CGRP mRNA in bone marrow by RT-PCR or in cells removed from the bone marrow and washed extensively in PBS (unpublished observations). Our failure to detect CGRP mRNA in hematopoietic cells in bone marrow is consistent with previous reports that find immunohistochemical staining of CGRP associated with nerve endings in bone marrow and not with hematopoietic cells [3 , 37 , 40 ]. This suggests that if cells in the lymphocyte lineages do produce CGRP as reported, they acquire this capability at later post-bone marrow stages of development. Thus, the data here infer that CGRP injected i.v. is mimicking the action of CGRP released from nerve endings in the bone marrow. In other areas of the body, CGRP is released from sensory nerve endings in response to inflammatory mediators, some of which are also known to up-regulate B cell development [5 ]. Thus, changes in these inflammatory mediators during an immune or inflammatory response could also lead to changes in CGRP release in bone marrow that could influence early B cell development.

The production of B cells is a finely regulated process that insures that the immune system acquires sufficient numbers of new B cells necessary to maintain efficient functioning of the immune system while avoiding potential negative consequences that would result from overproduction of new B cells. Naturally, this process requires positive and negative regulators. Although they play important roles, less effort has been directed toward understanding negative hematopoietic regulators. In the case of new B cells, overproduction could have negative consequences, such as failure to remove autoreactive clones, and unrestricted proliferation could increase the probability of developing lymphomas. Thus, negative regulation of B cell development by CGRP and other negative regulators is important in limiting the rate at which new B cells are produced. The accumulating evidence suggests that CGRP may have a fairly fluid and dynamic role in regulating early B cell development. Further studies using in vivo models will be necessary to better understand the role of CGRP and the sensory nervous system in regulating early B cell development.


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ACKNOWLEDGEMENTS
 
This research was supported by a grant from the National Institute of Neurological Disorders and Stroke, National Institutes of Health. We thank Ms. Jennifer Strange and Dr. Greg Bauman for assistance with the FACS analysis, Ms. Joella Grossoehme and Ms. Jennifer Olges for technical assistance, and Ms. Sarah Marks for editorial assistance.

Received March 30, 2006; revised October 24, 2006; accepted October 26, 2006.


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