Immunology 1991 72 451-452
ADONIS 001928059177A BRIEF COMMUNICATION
Stimulation of Ig production and growth of human lymphoblastoid B-cell lines by nerve growth factor H. KIMATA, A. YOSHIDA, C. ISHIOKA & H. MIKAWA Department of Pediatrics, Faculty of Medicine, Kyoto
University Hospital, Kyoto, Japan Acceptedfor publication 31 October 1990
SUMMARY The effects of nerve growth factor (NGF) on human lymphoblastoid B-cell lines were studied. NGF increased Ig production and proliferation by lymphoblastoid B-cell lines GM-1500, GM-1056 and CBL in a dose-dependent manner. As little as 0-01 ng/ml of NGF was effective. This effect was blocked by anti-NGF serum but not by control serum. Other cytokines, including interleukin (IL)1B, IL-2, IL-4, IL-5, interferon (IFN)-a, IFN-fl, IFN-y and granulocyte-macrophage colonystimulating factor (GM-CSF), did not stimulate Ig production. These results indicate that, in addition to its neurotropic effect NGF also acts as B-cell stimulatory factor. Nerve growth factor (NGF), a neurotrophic protein, has recently been shown to affect immunological and haematological responses. For example, NGF enhanced T-cell dependent antibody production in the rat.' In human, mouse NGF promoted haemopoietic colony growth and differentiation,2 stimulated B-cell and T-cell DNA synthesis, and enhanced IgM and IgA production in purified B cells.3 We have studied the effect of mouse NGF on Ig production and thymidine uptake by human lymphoblastoid B-cell lines, GM-1500, GM-1056 and CBL. GM- 1500 and GM- 1056 are IgG- and IgA-producing B-cell lines, respectively.4 They were obtained through NIGMS human genetic mutant cell repository (Camden, NJ). CBL#3 (CBL) is an EBV-transformed IgM-secreting lymphoblastoid line (a kind gift from Dr Christel H. Uittenbogaart, UCLA, Los Angeles, CA).' B cell lines were cultured (2 x 103/200 p1/well) with various concentrations of NGF (2 5S form of mouse NGF, a kind gift from Wako Pure Chemical, Ltd, Osaka, Japan), with or without sheep anti-NGF serum or control sheep serum, for 4 days in RPMI-1640 (M.A. Bioproducts, Walksville, MD) containing 10% foetal calf serum (FCS) (Irvine Scientific, Santa Ana, CA), 2 mm glutamine, 50 U/ml penicillin, and 50 yg/ml streptomycin. Sheep anti-NGF serum was generated by immunizing of sheep with the 2-5S form of mouse NGF together with Freund's complete adjuvant, and was kindly provided by Wako Pure Chemicals. The amount of Ig in the culture supernatants was measured by enzyme-linked immunoassay. Briefly, flatbottomed microtitre plates (Dynatech Laboratories Inc., Alexander, VA) were coated with anti-IgG (200 yg/ml; Tago Inc., Burlingame, CA), anti-IgA (400 ,g/ml; Tago) or anti-IgM (200 pg/ml; Tago). The plates were incubated for 1 hr at room Correspondence: Dr H. Kimata, Dept. of Pediatrics, Kyoto University Hospital, 54, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606,
temperature, washed and blocked with 1 % bovine serum
albumin-phosphate-buffered saline (BSA-PBS) for 30 min. After draining the plates, 10-25 p1 of supernatants, as well as standard curves of the appropriate purified 1g, were added, and 1% BSA-PBS was added to a final volume of 100 pl/well. After incubation for 2 hr at room temperature and washing thoroughly, affinity-purified isotyp-specific peroxidase-conjugated goat anti-human Ig (Kirkergaard & Perry Laboratories Inc., Gaithersburg, MD) diluted in 1% BSA-PBS was added for 1 hr. Thereafter the plates were allowed to react with 100 pi of 2-2 mM O-phenylenediamine (Nakarai Chemicals Ltd, Kyoto, Japan) containing 0-012% H202. The reaction was terminated with 100 p1 of 1-8 M H2SO4. Colour development was measured using an Autoreader (Model ELK 310; Bio-Tek Instrumental Inc., Winooski, VT). Cells were also pulsed with 1 pCi [3H]thymidine 8 h before harvest, and thymidine uptake measured.4 As shown in Fig. la, NGF enhanced Ig production from GM- 1500, GM- 1056 and CBL in a dose-dependent fashion, and as little as 0-01 ng/ml of NGF enhanced Ig production significantly. Similarly, as little as 0-01 ng/ml of NGF enhanced thymidine uptake (Fig. lb). NGF concentrations above 0-01 ng/ ml also increased cell numbers in these B-cell lines (data not shown). We did these experiments five times, and the results were identical. Specificity of the NGF effect is documented in Table 1. Enhancement of IgG production and thymidine uptake was blocked by anti-NGF serum but not by control sheep serum. Identical results were obtained in GM- 1056 and CBL (data not shown). We have previously reported that interleukin6 (IL-6) (100 U/ml) enhanced IgG production and thymidine uptake slightly by GM-1500.6 Thus the effect of IL-6 was compared to NGF. As shown in Table 1, enhancement of IgG production and thymidine uptake by IL-6 was completely blocked by anti-IL-6 serum but not by anti-NGF serum. Conversely, enhancement by NGF was not blocked by anti-IL-6
Japan.
451
452
H. Kimata et al. 22 20 18 16 c 4 ' 12
i
10
E
g
o
6 4 2 0
GM- 1500
GM-1056 CBL
5
(b)
O4
GM-1056
x
GM-1500
E
2
0
CBL
0
serum. We have also tested the effect of various cytokines. As we have previously reported, IL-IB (100 U/ml), IL-2 (100 U/ml), IL-4 (100 U/ml), interferon (IFN)-# (1000 U/ml), IFN-y (1000 U/ml), granulocyte-macrophage colony stimulating factor (GM-CSF) (100 pmol/l) or tumour necrosis factor (TNF)-a (100 pg/ml) did not enhance Ig production.6 In addition, IL-5 (1000 ng/ml) and IFN-cx (1000 U/ml) also failed to stimulate (data not shown). NGF at concentrations above 10 ng/ml has been shown to stimulate IgM production in pure B cells.3 In our B-cell line culture system, as little as 0-01 ng/ml enhanced both Ig (IgG, IgM and IgA) production and thymidine uptake. Therefore, NGF, in addition to its neurotropic effect, also may be able to act as B-cell stimulatory factor. It has been reported that in the central nervous system IL-6 induces NGF by astrocytes in viral diseases7 and activated B cell was found in multiple sclerosis.8 It is possible that NGF may play some roles in the B-cell stimulation in the central nervous system.
10 0-001 001 01 Conc. of NGF (ng/ml)
Figure 1. Effect of NGF on Ig production and thymidine uptake by GM1500, GM-1056, and CBL cells. B-cell lines were cultured with various concentrations of NGF. IgG (0), IgA (A) and IgM (0) production (a) and thymidine uptake (b) were measured after 4 days. Values are the mean + 1 SD from triplicate cultures.
ACKNOWLEDGMENTS We thank Wako Pure Chemical Industries Ltd, for providing NGF and anti-NGF serum. This work was supported by a grant from the Ministry of Health and Welfare, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
REFERENCES
Table 1. Specificity of the effect of NGF
Thymidine Factors
Medium NGF NGF + anti-NGF serum NGF+ control serum NGF + anti-IL-6 serum IL-6 IL-6+ anti-IL-6 serum IL-6 + anti-NGF serum
IgG (ng/ml)
(x 10-3)
2-1+0-2 20-3+19 2-2 +0-2 20-2+ 1-3 20-4+ 1-8 28±+0 3 2-0 + 0-2 2-9+0 3
1-4+0-1 45+03 1-3 + 01 4-6+004 4-5 +0 3 2-1+0-2 1-5 + 01 3-3 +0 3
GM-1500 cells were cultured with indicated factors for 4 days. NGF was used at 1 ng/ml, anti-NGF serum at 1:100, control serum at 1:100, IL-6 at 100 U/ml and anti-IL-6 serum at 20 Mg/ml. Results are expressed as mean + 1 SD of triplicate cultures.
1. MANNING P.T., PUSSEL L.H., SIMMONS B. & JOHNSON E.M. (1985) Protection from guanethidine-induced neuronal destruction by nerve growth factor: effects of NGF on immune function. Brain Res. 340, 61. 2. MASUDA H., COUGHLIN M.D., BIENENSTOCK J. & DENBURG J.A. (1988) Nerve growth factor promotes human hemopoietic colony growth and differentiation. Proc. natl. Acad. Sci. U.S.A. 85, 6508. 3. OTTEN U., EHRHARD P. & PECK R. (1989) Nerve growth factor induces growth and differentiation of human B lymphocytes. Proc. natl. Acad. Sci. U.S.A. 86, 10059. 4. KIMATA H. & SAXON A. (1988) Subset of natural killer cells is induced by immune complexes to display Fc receptors for IgE and IgA and demonstrate isotype regulatory function. J. clin. Invest. 82, 160. 5. KANOWITH-KLEIN S., SAXON A. & UITTENBOGAART C.H. (1987) Constitutive production of B cell differentiation factor-like activity by human T and B cell lines. Eur. J. Immunol. 17, 593. 6. KIMATA H., SHERR, E.H. & SAXON A. (1988) Human natural killer (NK) cells produce a late-acting B-cell differentiation activity. J. clin. Immunol. 8, 381. 7. FREI K., MALIPIERO U.V., LEIST T.P., ZINKERNAGEL R.M., SCHAMB M.E. & FONTANA A. (1989) On the cellular source and function of interleukin 6 produced in the central nervous system in viral diseases. Eur. J. Immunol. 19, 689. 8. TOURTELLOTTE W.W. & MA B.I. (1978) Multiple sclerosis: The blood-brain-barrier and the measurement of de novo central nervous system IgG synthesis. Neurology, 28, 76.
ADONIS 001928059177A BRIEF COMMUNICATION
Stimulation of Ig production and growth of human lymphoblastoid B-cell lines by nerve growth factor H. KIMATA, A. YOSHIDA, C. ISHIOKA & H. MIKAWA Department of Pediatrics, Faculty of Medicine, Kyoto
University Hospital, Kyoto, Japan Acceptedfor publication 31 October 1990
SUMMARY The effects of nerve growth factor (NGF) on human lymphoblastoid B-cell lines were studied. NGF increased Ig production and proliferation by lymphoblastoid B-cell lines GM-1500, GM-1056 and CBL in a dose-dependent manner. As little as 0-01 ng/ml of NGF was effective. This effect was blocked by anti-NGF serum but not by control serum. Other cytokines, including interleukin (IL)1B, IL-2, IL-4, IL-5, interferon (IFN)-a, IFN-fl, IFN-y and granulocyte-macrophage colonystimulating factor (GM-CSF), did not stimulate Ig production. These results indicate that, in addition to its neurotropic effect NGF also acts as B-cell stimulatory factor. Nerve growth factor (NGF), a neurotrophic protein, has recently been shown to affect immunological and haematological responses. For example, NGF enhanced T-cell dependent antibody production in the rat.' In human, mouse NGF promoted haemopoietic colony growth and differentiation,2 stimulated B-cell and T-cell DNA synthesis, and enhanced IgM and IgA production in purified B cells.3 We have studied the effect of mouse NGF on Ig production and thymidine uptake by human lymphoblastoid B-cell lines, GM-1500, GM-1056 and CBL. GM- 1500 and GM- 1056 are IgG- and IgA-producing B-cell lines, respectively.4 They were obtained through NIGMS human genetic mutant cell repository (Camden, NJ). CBL#3 (CBL) is an EBV-transformed IgM-secreting lymphoblastoid line (a kind gift from Dr Christel H. Uittenbogaart, UCLA, Los Angeles, CA).' B cell lines were cultured (2 x 103/200 p1/well) with various concentrations of NGF (2 5S form of mouse NGF, a kind gift from Wako Pure Chemical, Ltd, Osaka, Japan), with or without sheep anti-NGF serum or control sheep serum, for 4 days in RPMI-1640 (M.A. Bioproducts, Walksville, MD) containing 10% foetal calf serum (FCS) (Irvine Scientific, Santa Ana, CA), 2 mm glutamine, 50 U/ml penicillin, and 50 yg/ml streptomycin. Sheep anti-NGF serum was generated by immunizing of sheep with the 2-5S form of mouse NGF together with Freund's complete adjuvant, and was kindly provided by Wako Pure Chemicals. The amount of Ig in the culture supernatants was measured by enzyme-linked immunoassay. Briefly, flatbottomed microtitre plates (Dynatech Laboratories Inc., Alexander, VA) were coated with anti-IgG (200 yg/ml; Tago Inc., Burlingame, CA), anti-IgA (400 ,g/ml; Tago) or anti-IgM (200 pg/ml; Tago). The plates were incubated for 1 hr at room Correspondence: Dr H. Kimata, Dept. of Pediatrics, Kyoto University Hospital, 54, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606,
temperature, washed and blocked with 1 % bovine serum
albumin-phosphate-buffered saline (BSA-PBS) for 30 min. After draining the plates, 10-25 p1 of supernatants, as well as standard curves of the appropriate purified 1g, were added, and 1% BSA-PBS was added to a final volume of 100 pl/well. After incubation for 2 hr at room temperature and washing thoroughly, affinity-purified isotyp-specific peroxidase-conjugated goat anti-human Ig (Kirkergaard & Perry Laboratories Inc., Gaithersburg, MD) diluted in 1% BSA-PBS was added for 1 hr. Thereafter the plates were allowed to react with 100 pi of 2-2 mM O-phenylenediamine (Nakarai Chemicals Ltd, Kyoto, Japan) containing 0-012% H202. The reaction was terminated with 100 p1 of 1-8 M H2SO4. Colour development was measured using an Autoreader (Model ELK 310; Bio-Tek Instrumental Inc., Winooski, VT). Cells were also pulsed with 1 pCi [3H]thymidine 8 h before harvest, and thymidine uptake measured.4 As shown in Fig. la, NGF enhanced Ig production from GM- 1500, GM- 1056 and CBL in a dose-dependent fashion, and as little as 0-01 ng/ml of NGF enhanced Ig production significantly. Similarly, as little as 0-01 ng/ml of NGF enhanced thymidine uptake (Fig. lb). NGF concentrations above 0-01 ng/ ml also increased cell numbers in these B-cell lines (data not shown). We did these experiments five times, and the results were identical. Specificity of the NGF effect is documented in Table 1. Enhancement of IgG production and thymidine uptake was blocked by anti-NGF serum but not by control sheep serum. Identical results were obtained in GM- 1056 and CBL (data not shown). We have previously reported that interleukin6 (IL-6) (100 U/ml) enhanced IgG production and thymidine uptake slightly by GM-1500.6 Thus the effect of IL-6 was compared to NGF. As shown in Table 1, enhancement of IgG production and thymidine uptake by IL-6 was completely blocked by anti-IL-6 serum but not by anti-NGF serum. Conversely, enhancement by NGF was not blocked by anti-IL-6
Japan.
451
452
H. Kimata et al. 22 20 18 16 c 4 ' 12
i
10
E
g
o
6 4 2 0
GM- 1500
GM-1056 CBL
5
(b)
O4
GM-1056
x
GM-1500
E
2
0
CBL
0
serum. We have also tested the effect of various cytokines. As we have previously reported, IL-IB (100 U/ml), IL-2 (100 U/ml), IL-4 (100 U/ml), interferon (IFN)-# (1000 U/ml), IFN-y (1000 U/ml), granulocyte-macrophage colony stimulating factor (GM-CSF) (100 pmol/l) or tumour necrosis factor (TNF)-a (100 pg/ml) did not enhance Ig production.6 In addition, IL-5 (1000 ng/ml) and IFN-cx (1000 U/ml) also failed to stimulate (data not shown). NGF at concentrations above 10 ng/ml has been shown to stimulate IgM production in pure B cells.3 In our B-cell line culture system, as little as 0-01 ng/ml enhanced both Ig (IgG, IgM and IgA) production and thymidine uptake. Therefore, NGF, in addition to its neurotropic effect, also may be able to act as B-cell stimulatory factor. It has been reported that in the central nervous system IL-6 induces NGF by astrocytes in viral diseases7 and activated B cell was found in multiple sclerosis.8 It is possible that NGF may play some roles in the B-cell stimulation in the central nervous system.
10 0-001 001 01 Conc. of NGF (ng/ml)
Figure 1. Effect of NGF on Ig production and thymidine uptake by GM1500, GM-1056, and CBL cells. B-cell lines were cultured with various concentrations of NGF. IgG (0), IgA (A) and IgM (0) production (a) and thymidine uptake (b) were measured after 4 days. Values are the mean + 1 SD from triplicate cultures.
ACKNOWLEDGMENTS We thank Wako Pure Chemical Industries Ltd, for providing NGF and anti-NGF serum. This work was supported by a grant from the Ministry of Health and Welfare, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
REFERENCES
Table 1. Specificity of the effect of NGF
Thymidine Factors
Medium NGF NGF + anti-NGF serum NGF+ control serum NGF + anti-IL-6 serum IL-6 IL-6+ anti-IL-6 serum IL-6 + anti-NGF serum
IgG (ng/ml)
(x 10-3)
2-1+0-2 20-3+19 2-2 +0-2 20-2+ 1-3 20-4+ 1-8 28±+0 3 2-0 + 0-2 2-9+0 3
1-4+0-1 45+03 1-3 + 01 4-6+004 4-5 +0 3 2-1+0-2 1-5 + 01 3-3 +0 3
GM-1500 cells were cultured with indicated factors for 4 days. NGF was used at 1 ng/ml, anti-NGF serum at 1:100, control serum at 1:100, IL-6 at 100 U/ml and anti-IL-6 serum at 20 Mg/ml. Results are expressed as mean + 1 SD of triplicate cultures.
1. MANNING P.T., PUSSEL L.H., SIMMONS B. & JOHNSON E.M. (1985) Protection from guanethidine-induced neuronal destruction by nerve growth factor: effects of NGF on immune function. Brain Res. 340, 61. 2. MASUDA H., COUGHLIN M.D., BIENENSTOCK J. & DENBURG J.A. (1988) Nerve growth factor promotes human hemopoietic colony growth and differentiation. Proc. natl. Acad. Sci. U.S.A. 85, 6508. 3. OTTEN U., EHRHARD P. & PECK R. (1989) Nerve growth factor induces growth and differentiation of human B lymphocytes. Proc. natl. Acad. Sci. U.S.A. 86, 10059. 4. KIMATA H. & SAXON A. (1988) Subset of natural killer cells is induced by immune complexes to display Fc receptors for IgE and IgA and demonstrate isotype regulatory function. J. clin. Invest. 82, 160. 5. KANOWITH-KLEIN S., SAXON A. & UITTENBOGAART C.H. (1987) Constitutive production of B cell differentiation factor-like activity by human T and B cell lines. Eur. J. Immunol. 17, 593. 6. KIMATA H., SHERR, E.H. & SAXON A. (1988) Human natural killer (NK) cells produce a late-acting B-cell differentiation activity. J. clin. Immunol. 8, 381. 7. FREI K., MALIPIERO U.V., LEIST T.P., ZINKERNAGEL R.M., SCHAMB M.E. & FONTANA A. (1989) On the cellular source and function of interleukin 6 produced in the central nervous system in viral diseases. Eur. J. Immunol. 19, 689. 8. TOURTELLOTTE W.W. & MA B.I. (1978) Multiple sclerosis: The blood-brain-barrier and the measurement of de novo central nervous system IgG synthesis. Neurology, 28, 76.
