To the Editor:

Several studies on the differentiation, function, and phenotype of activated B lymphocytes are currently using an in vitro system in which CD40 stimulation of B cells is induced by CD40 ligand (CD40L) expressed on the surface of fibroblasts, usually mouse L cells transfected with the CD40L gene.1 The ligation of CD40 on B cells by the CD40L, which is expressed under physiological conditions on activated γδ, CD4+ αβ T, or on mast cells and basophils, plays a crucial role in the induction and regulation of a T-cell–dependent humoral immune response.2,3 The CD40/CD40L interaction costimulates B-cell proliferation and differentiation into memory B cells.4 5 Production of Igs and isotype class switch are also regulated by T-B cell interaction through the CD40/CD40L pathway. In addition, the role of CD40 ligation in B-cell terminal differentiation is also being actively studied.

CD40 is detected on other professional antigen-presenting cells (APC) such as thymic epithelial cells, activated macrophages, dendritic cells (DC), follicular dendritic cells (FDC), and also on activated fibroblasts, vascular epithelial, or glia cells.3 Ligation of CD40 on macrophages, DCs, or epithelial cells induces activation, proliferation, and the production of various cytokines including interleukin-12 (IL-12), which is responsible for the development of Th1 responses.2 CD40 stimulation of monocytes by fibroblastoid murine L cells or monkey CV-1/EBNA kidney cells expressing CD40L resulted in the induction of different sets of cytokines.6,7 These differences were attributed to (1) the variable expression of CD40L, (2) the distinct costimulatory molecules on the cell membrane, and/or (3) the secretion of various costimulatory cytokines by these cells.3 Experiments monitoring the expression of adhesion molecules on Langerhans cells upon activation by soluble CD40L or by CD40L expressed on the surface of L cells also gave rise to contradictory data.7 

The CD40 molecule belongs to the family of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptors, including CD27, Fas/APO-1, and the p75 low-affinity receptor for NGF. These molecules are involved in regulation of cell proliferation and programmed cell death, mainly in immune cells. Indeed, in addition to its neurotrophic activity, NGF has also been suggested to play a regulatory role on the cells of the immune system, and many studies have been focused on the activity of NGF on B lymphocytes.8 The NGF has been found to be an autocrine survival factor for memory B cells and to be active in regulating Ig production by B cells.9,10 Helper T lymphocytes of both the Th1 and Th2 type were shown to produce NGF,11 12 suggesting a role for NGF in modulating immune responses.

We have tested the cell culture supernatant of irradiated L cells and CD40L-transfected L cells for the presence of soluble NGF by a sensitive quantitative enzyme-linked immunoassay (ELISA). The cells were cultured in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum and antibiotics. After irradiation (150 Gy) the cells were cultured in serum-free AIM-V medium at 4 × 105 cells/mL and supernatants were obtained after 24 hours. The ELISA for NGF detection was performed according to the manufacturer’s instructions as follows: polystyrene 96-well immunoplates (Nunc, Roskilde, Denmark) were coated for 2 hours at 37°C with 100 μL of 0.15 μg/mL of mouse monoclonal antibody (MoAb) 27/21 specific for NGF (Boehringer Mannheim, Mannheim, Germany) diluted in 0.05 mol/L carbonate buffer (pH 9.6). The plates were further incubated with the blocking solution (0.5% bovine serum albumin in coating buffer) for 30 minutes at 37°C. Cell culture supernatants and the NGF standard curve diluted in the same culture medium were incubated overnight at 4°C. The presence of bound NGF was detected by 4-hour incubation at 37°C with 4 mU/well of anti-NGF antibody 27/21 conjugated with β-galactosidase (Boehringer Mannheim). The plates were then incubated overnight at 37°C with 200 μL of chlorophenolred-β-D-galactopyranoside (2 mg/mL) in substrate buffer and the optical density was monitored at 575 nm using an Elisa Processor II (Behringwerke AG, Marburg, Germany). All the samples and the NGF standard curve were run in triplicate wells. As summarized in Table 1, we found that irradiated L cells and CD40L-transfected L cells released a considerable amount of NGF, and comparable amounts of NGF also were detected in the supernatant of irradiated A9 fibrosarcoma cells, another mouse fibroblastoid cell line.

As mentioned above, one aspect of B-cell function that has been studied using the CD40/CD40L system is the requirement of CD40 ligation for differentiation of B cells into antibody-secreting plasma cells. Several reports have indicated that CD40 signaling promotes differentiation of B cells,13 although it is still debated if CD40 engagement is fundamental for B-cell terminal differentiation.5 In fact, naive and memory B cells were shown to respond differently to CD40 signaling.13 Since memory B lymphocytes have been shown to rely on secreted NGF for survival, we reasoned that NGF produced in the CD40 system may also sustain survival of these cells and increase the number of terminally differentiated B cells in the culture. We analyzed IgG secretion by human peripheral blood memory B lymphocytes cocultured with CD40L-expressing L cells in presence of IL-2 and IL-10.13As shown in Fig 1A, detectable NGF concentration in coculture supernatants after 5 days of incubation was decreased in presence of a neutralizing anti-NGF MoAb. Neutralization of NGF induced a 38% to 41% reduction in IgG production by memory B cells from two donors cocultured with CD40L-transfected L cells (Fig 1B and C).

Fig. 1.

(A) The effect of anti-NGF MoAb on NGF detection in coculture supernatants of memory B cells cocultured with CD40L-transfected L cells. (B [donor 1] and C [donor 2]) The mean IgG production by memory B cells cocultured with CD40L-transfected L cells. B lymphocytes were purified from buffy coats by using anti-CD19 M450 Dynabeads (Dynal, Oslo, Norway), and memory CD19+ B cells were isolated by negative selection using a rabbit anti-human IgD antibody and anti-rabbit IgG antibody coated microbeads (Miltenyi Biotec, Bergisch, Germany). Memory B cells were cocultured with CD40L-expressing L cells (B cells: fibroblasts, 5:1) in IMDM containing IL-2 (20 U/mL) and IL-10 (10 ng/mL), in the presence of a neutralizing anti-NGF MoAb (15 μg/mL; R & D Systems, Minneapolis, MN) or negative mouse IgG (15 μg/mL). Cultures were performed in six parallel wells containing 1 × 105 B cells. Cell-culture supernatants were collected after 5 days and tested for NGF or IgG content by capture ELISA.19 SEM were <10% for the NGF ELISA and <15% for the IgG ELISA. *P < .05 and **P< .005 by t-test.

Fig. 1.

(A) The effect of anti-NGF MoAb on NGF detection in coculture supernatants of memory B cells cocultured with CD40L-transfected L cells. (B [donor 1] and C [donor 2]) The mean IgG production by memory B cells cocultured with CD40L-transfected L cells. B lymphocytes were purified from buffy coats by using anti-CD19 M450 Dynabeads (Dynal, Oslo, Norway), and memory CD19+ B cells were isolated by negative selection using a rabbit anti-human IgD antibody and anti-rabbit IgG antibody coated microbeads (Miltenyi Biotec, Bergisch, Germany). Memory B cells were cocultured with CD40L-expressing L cells (B cells: fibroblasts, 5:1) in IMDM containing IL-2 (20 U/mL) and IL-10 (10 ng/mL), in the presence of a neutralizing anti-NGF MoAb (15 μg/mL; R & D Systems, Minneapolis, MN) or negative mouse IgG (15 μg/mL). Cultures were performed in six parallel wells containing 1 × 105 B cells. Cell-culture supernatants were collected after 5 days and tested for NGF or IgG content by capture ELISA.19 SEM were <10% for the NGF ELISA and <15% for the IgG ELISA. *P < .05 and **P< .005 by t-test.

Close modal

In previous studies, murine L cells were shown to secrete macrophage colony-stimulatory factor (CSF-1) and both mouse and human fibroblasts have been found to release biologically active NGF to the culture medium.14 At the time NGF release from mouse and human fibroblasts was first described, the CD40L-transfected L cells were not yet being used as a model system and it was not known that NGF could function as an immunoregulatory factor on B cells.8-12 Over the years, many studies using CD40L-transfected L cells have been performed on B lymphocytes and on other cell types. Both the p140TrkA and the p75 NGF receptors are expressed in resting B cells and are upregulated upon activation.15 Because murine and human NGF show high homology in their structure and biological function,16,17 we would like to draw attention to the effects of murine NGF released by CD40L-transfected L cells on human NGF receptors, especially when this system is used to study B-cell function and differentiation, as reported here. Appropriate negative controls for such experiments should be carefully included to rule out any interference of NGF on the functional and phenotypic characteristics of the target cells tested by using this in vitro model. In addition, activated monocytes also express the high affinity NGF receptor,18 and the role of NGF should also be considered in the interpretation of controversial results obtained with monocytes and DCs activated by CD40L expressed on different carrier cells or used as a soluble protein.

Supported by the Swedish Medical Research Council, the Hedlunds Foundation, and the Foundation Blanceflor Boncompagni-Ludovisi.

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