CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
60,
145-151 (1991)
BRIEF COMMUNICATION Nerve Growth Factor Inhibits lmmunoglobulin Production but Not Proliferation of Human Plasma Cell Lines HAJIME KIMATA, AKIRA YOSHIDA, CHIHIRO ISHIOKA, AND HARUKI Department
of Pediatrics,
Faculty
of Medicine,
Kyoto
University
Hospital,
Kyoto,
by
MIKAWA Japan
The effects of nerve growth factor (NGF) on human plasma cells were studied. NGF inhibited immunoglobulin (Ig) production but not thymidine uptake by human plasma cell lines IM-9 and AF-10 in a dose-dependent fashion. This NGF-induced inhibition of Ig production was specific. since inhibition was blocked by anti-NGF serum but not by control serum. Interleukin (IL)-6 did not affect Ig production by IM-9 and AF-10; however. IL-6 restored NGF-induced inhibition of Ig production. NGF also inhibited Ig production (IgG, IgM. and IgA) without affecting thymidine uptake by PCA-1 + plasma cells generated in vitro. This inhibition was also blocked by anti-NGF serum but not by control serum and was restored by IL-6. These results suggest that NGF may interact with IL-6 in control of Ig production by plasma cells. $0 1991 Academic press. II-K.
INTRODUCTION
Nerve growth factor (NGF) is a neurotrophic protein with greater than 90% identity in the amino acid sequence between the murine and human forms (1). Recently, NGF has been shown to affect immunological and hematological responses. In rats, NGF was found to enhance T-cell-dependent antibody production (2) and in humans, NGF was found to promote hemopoietic colony growth and differentiation (3). NGF also stimulates B-cell DNA synthesis and enhances IgM and IgA production in purified B cells (4). We have previously reported that NGF specifically induces IgG4 production by purified B cells, especially by large activated B cells (5). While studying B-cell responses to NGF we noticed that NGF inhibited Ig production without affecting thymidine uptake by human plasma cell lines IM-9 and AF-10, and PCA-I+ plasma cells generated in vitro. The relationship of NGF to other plasma cell stimulatory cytokines will be discussed. MATERIALS
AND METHODS
Reagents The following factors or cytokines were kindly provided by each company or institute as indicated: purified mouse NGF (2.5s form) and sheep anti-NGF serum (Wako Pure Chemical Industries, Ltd., Osaka, Japan), human recombinant interleukin (IL)-5 (Suntory Research Center, Osaka), and human recombinant IL-6 and rabbit anti-IL-6 IgG antibody (kind gifts from Drs. T. Hirano and T. Kishimoto, Institute for Molecular and Cellular Biology, Osaka). Human recombinant IL-3 was obtained from Genzyme (Boston, MA). Normal sheep serum and rabbit IgG fraction from normal serum were purchased from Nakalai Chemicals (Kyoto, 145 0090-1229/91 $1.50 Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
146
BRIEF
COMMUNICATION
Japan). The culture medium (abbreviated as CRPMI) was RPM1 1640 (M.A. Bioproducts, Walksville, MD) containing 10% FCS (Irvine Scientific), 2 m&f glutamine, 50 U/ml penicillin, and 50 p&ml streptomycin.
Plasma cell lines. The IgG-producing IM-9 cell line was obtained from the Japanese Cancer Research Resources Bank (6). The IgE-producing AF-10 (a kind gift from Dr. Andrew Saxon, UCLA) is a subclone of U266 kindly provided by Dr. Kenneth Nilsson (Laboratory of Tumor Biology, University Hospital, Uppsala, Sweden) (7). The generation and purification of plasma cells in vitro were carried out as follows. Tonsils (kind gifts from Dr. K. Nagahara, National Kyoto Hospital, Kyoto, Japan) obtained at tonsillectomy were disrupted, and mononuclear cells were obtained by buoyant density centrifugation on a Ficoll-Hypaque gradient (7). Mononuclear cells (2 x 106/ml) were cultured with 10 p&ml of pokeweed mitogen (GIBCO, Grand Island, NY) for 7 days in CRPMI at 37°C with 5% CO2 in air and then harvested. Since activated T cells and monocytes also express the PCA-1 antigen (8), cultured cells were depleted of T cells using the SRBC rosette technique, and monocytes were depleted using L-leucine methyl ester preincubation methods (9). Thereafter, PCA-1 + cells were purified by panning methods using the anti-PCA-1 antibody (Coulter Immunology, Hialeah, FL) as previously described (7). This cell fraction contained >98% plasmablast-plasma cells as demonstrated by Giemsa staining. Cell Cultures
IM-9 and AF-10 cells were cultured (1 x lo3 cells/200 pi/well) in %-well Ubottomed microtiter plates (Coster, Cambridge, MA) for 4 days with or without NGF or other factors, and then pulsed with 1 pCi r3H]thymidine for 8 hr before harvest. The amount of Ig in supematants was measured by ELISA, and thymidine uptake was simultaneously determined. Net Ig production was calculated by subtracting the values of Ig obtained in parallel cultures containing cycloheximide (100 pg/ml) (7). In some experiments, viable cell numbers were counted by trypan blue exclusion. Purified PCA-1 + cells (2 x lo4 cells/200 pi/well) were cultured with or without NGF or other factors for 5 days, and Ig production in supematants was measured by ELISA. PCA- It cells were also cultured for 2 days and pulsed as above for 16 hr before harvest, and their thymidine uptake was determined. Statistical analysis was performed with the two-tailed Student’s t test. RESULTS
NGF was found to inhibit Ig production by IM-9 and AF-10 ceIIs in a dosedependent fashion (Fig. 1A). In contrast, NGF did not affect cell growth as determined by thymidine uptake or ceil numbers at any of the concentrations tested (Fig. IB). The low amount of Ig production by IM-9 or AF-10 was not preformed cytoplasmic Ig since the addition of cycloheximide completely blocked Ig production by these cell lines as previously described (7). The kinetics of control net IgG production (&ml) by IM-9 was 6.2 -+ 0.6 (Day I), 24.5 -+ 2.3 (Day 2), 45.9 + 5.1 (Day 3), and 65.2 -t- 5.4 (Day 4) while control net IgE production (ng/mI) by
147
BRIEF COMMUNICATION mc
(A)
80
-
0
ol
;
' 0
I
I
I
I
I
0.01
0.1
1
5
10
JO
8-(B)
-8
a
-4
n X zi .-t 0
E > f:
*” @-+---+~-~-~-~ 2-
Ilk-9 AF-10
T
; 2 2 ;I 5 0
I I 1 1 1 0 0.01 0.1 1 5 10 Concentrations of NGF (rig/ml) FIG. 1. Effect of NGF on Ig production and cell growth by IM-9 and AF-10 cells. IM-9 (.A=) and AF-10 cells (OAU) were cultured with increasing concentrations of NGF for 4 days. IgG (0) and IgE production (0) in (A) and thymidine uptake (AA) and cell number (mEI) in (B) were determined. Vahtes are the means ? 1 SD of triplicate cultures. *Significant inhibition of Ig production (P < 0.01 compared to medium).
0
I
0
AF-10 was 3.2 + 0.6 (Day I), 17.0 + 0.7 (Day 2), 32.2 + 2.8 (Day 3), and 45.2 t 4.1 (Day 4). NGF did not inhibit Ig production by IM-9 or AF-10 when NGF was added to the culture after 1 day or when NGF was added to the culture at the initiation of culture then removed after l-3 days (data not shown). These results indicate that NGF must be added at the initiation of culture and must be present throughout the entire culture period. The specificity of the NGF effect was documented in Fig 2. NGF-induced inhibition of Ig production by IM-9 and AF-10 was blocked by anti-NGF serum but not by control serum. It has been reported that IL-3, IL-4, IL-S, and IL-6 affect plasma cell responses (6, 10, 11). However, IL-3 (0.1-10 U/ml), IL-4 (0.1-10 U/ml), IL-5 (0. I-10 &ml), and IL-6 (0. f-10 U/ml) did not affect Ig production or thymidine uptake by IM-9 or AF-IO (data not shown). In contrast, the addition of IL-6 restored the NGF-induced inhibition of Ig production while that of IL-3,
148
BRIEF COMMUNICATION IMP 9
t
AFmmIO
Medium
t-c
NGF NGF +anti-NGF NGF+ control
serum
NGF+ IL- 3 NGF+ IL-4 NGF+IL-
5
NGF +IL-
6 0
20
40
60
0
20
40
60
80
IgE Production (rig/ml) IgG Production (rig/ml) FIG. 2. Specificity of the NGF effect. IM-9 (W) and AF-IO cells (0) were cultured with the indicated factors. NGF was used at 5 &ml, sheep anti-NGF serum at I: 100, control sheep serum at 1: 100, IL-3 at 10 U/ml, IL-4 at 10 U/ml, IL-5 at IO &ml, and IL-6 at IO U/ml. Values are the means + I SD of triplicate cultures. *Significant inhibition of Ig production (P < 0.01 compared to medium).
IL-4, or IL-5 had no effect on the NGF-induced inhibition of Ig production (Fig. 2). The dose-response pattern of the IL-6 addition on NGF-induced Ig inhibition is shown in Table 1. As little as 1 U/ml of IL-6 significantly restored NGF-induced inhibition of Ig production, and inhibition was completely restored at a dose of 10 TABLE EFFECT
Factors Medium NGF NGF + IL-6 (0.1 U/ml) NGF + IL-6 (I U/ml) NGF + IL-6 (10 U/ml) NGF + IL-6 (IO U/ml) + anti-IL-6 antibody NGF + IL-6 (10 U/ml) + control antibody
OF IL-6
ON NGF-INDUCED
I INHIBITION
OF
Ig PRODUCTION
IM-9
AF-IO
IgG (rig/ml)
I@ (rig/ml)
56.6 23.2 24.8 39.4 55.8
(59)a,b (56)b (30)b (1)
45.3 18.1 19.5 30.4 44.6
(6O)b (57)b (33)b (2)
25.6 (55)b
19.0 (58)b
55.7 (2)
45.1 (0)
NOW. NGF was used at 5 r&ml, anti-IL-6 antibody at 2 &ml, and control antibody at 2 &ml. Values are the means of triplicate cultures. SD were
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
60,
145-151 (1991)
BRIEF COMMUNICATION Nerve Growth Factor Inhibits lmmunoglobulin Production but Not Proliferation of Human Plasma Cell Lines HAJIME KIMATA, AKIRA YOSHIDA, CHIHIRO ISHIOKA, AND HARUKI Department
of Pediatrics,
Faculty
of Medicine,
Kyoto
University
Hospital,
Kyoto,
by
MIKAWA Japan
The effects of nerve growth factor (NGF) on human plasma cells were studied. NGF inhibited immunoglobulin (Ig) production but not thymidine uptake by human plasma cell lines IM-9 and AF-10 in a dose-dependent fashion. This NGF-induced inhibition of Ig production was specific. since inhibition was blocked by anti-NGF serum but not by control serum. Interleukin (IL)-6 did not affect Ig production by IM-9 and AF-10; however. IL-6 restored NGF-induced inhibition of Ig production. NGF also inhibited Ig production (IgG, IgM. and IgA) without affecting thymidine uptake by PCA-1 + plasma cells generated in vitro. This inhibition was also blocked by anti-NGF serum but not by control serum and was restored by IL-6. These results suggest that NGF may interact with IL-6 in control of Ig production by plasma cells. $0 1991 Academic press. II-K.
INTRODUCTION
Nerve growth factor (NGF) is a neurotrophic protein with greater than 90% identity in the amino acid sequence between the murine and human forms (1). Recently, NGF has been shown to affect immunological and hematological responses. In rats, NGF was found to enhance T-cell-dependent antibody production (2) and in humans, NGF was found to promote hemopoietic colony growth and differentiation (3). NGF also stimulates B-cell DNA synthesis and enhances IgM and IgA production in purified B cells (4). We have previously reported that NGF specifically induces IgG4 production by purified B cells, especially by large activated B cells (5). While studying B-cell responses to NGF we noticed that NGF inhibited Ig production without affecting thymidine uptake by human plasma cell lines IM-9 and AF-10, and PCA-I+ plasma cells generated in vitro. The relationship of NGF to other plasma cell stimulatory cytokines will be discussed. MATERIALS
AND METHODS
Reagents The following factors or cytokines were kindly provided by each company or institute as indicated: purified mouse NGF (2.5s form) and sheep anti-NGF serum (Wako Pure Chemical Industries, Ltd., Osaka, Japan), human recombinant interleukin (IL)-5 (Suntory Research Center, Osaka), and human recombinant IL-6 and rabbit anti-IL-6 IgG antibody (kind gifts from Drs. T. Hirano and T. Kishimoto, Institute for Molecular and Cellular Biology, Osaka). Human recombinant IL-3 was obtained from Genzyme (Boston, MA). Normal sheep serum and rabbit IgG fraction from normal serum were purchased from Nakalai Chemicals (Kyoto, 145 0090-1229/91 $1.50 Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
146
BRIEF
COMMUNICATION
Japan). The culture medium (abbreviated as CRPMI) was RPM1 1640 (M.A. Bioproducts, Walksville, MD) containing 10% FCS (Irvine Scientific), 2 m&f glutamine, 50 U/ml penicillin, and 50 p&ml streptomycin.
Plasma cell lines. The IgG-producing IM-9 cell line was obtained from the Japanese Cancer Research Resources Bank (6). The IgE-producing AF-10 (a kind gift from Dr. Andrew Saxon, UCLA) is a subclone of U266 kindly provided by Dr. Kenneth Nilsson (Laboratory of Tumor Biology, University Hospital, Uppsala, Sweden) (7). The generation and purification of plasma cells in vitro were carried out as follows. Tonsils (kind gifts from Dr. K. Nagahara, National Kyoto Hospital, Kyoto, Japan) obtained at tonsillectomy were disrupted, and mononuclear cells were obtained by buoyant density centrifugation on a Ficoll-Hypaque gradient (7). Mononuclear cells (2 x 106/ml) were cultured with 10 p&ml of pokeweed mitogen (GIBCO, Grand Island, NY) for 7 days in CRPMI at 37°C with 5% CO2 in air and then harvested. Since activated T cells and monocytes also express the PCA-1 antigen (8), cultured cells were depleted of T cells using the SRBC rosette technique, and monocytes were depleted using L-leucine methyl ester preincubation methods (9). Thereafter, PCA-1 + cells were purified by panning methods using the anti-PCA-1 antibody (Coulter Immunology, Hialeah, FL) as previously described (7). This cell fraction contained >98% plasmablast-plasma cells as demonstrated by Giemsa staining. Cell Cultures
IM-9 and AF-10 cells were cultured (1 x lo3 cells/200 pi/well) in %-well Ubottomed microtiter plates (Coster, Cambridge, MA) for 4 days with or without NGF or other factors, and then pulsed with 1 pCi r3H]thymidine for 8 hr before harvest. The amount of Ig in supematants was measured by ELISA, and thymidine uptake was simultaneously determined. Net Ig production was calculated by subtracting the values of Ig obtained in parallel cultures containing cycloheximide (100 pg/ml) (7). In some experiments, viable cell numbers were counted by trypan blue exclusion. Purified PCA-1 + cells (2 x lo4 cells/200 pi/well) were cultured with or without NGF or other factors for 5 days, and Ig production in supematants was measured by ELISA. PCA- It cells were also cultured for 2 days and pulsed as above for 16 hr before harvest, and their thymidine uptake was determined. Statistical analysis was performed with the two-tailed Student’s t test. RESULTS
NGF was found to inhibit Ig production by IM-9 and AF-10 ceIIs in a dosedependent fashion (Fig. 1A). In contrast, NGF did not affect cell growth as determined by thymidine uptake or ceil numbers at any of the concentrations tested (Fig. IB). The low amount of Ig production by IM-9 or AF-10 was not preformed cytoplasmic Ig since the addition of cycloheximide completely blocked Ig production by these cell lines as previously described (7). The kinetics of control net IgG production (&ml) by IM-9 was 6.2 -+ 0.6 (Day I), 24.5 -+ 2.3 (Day 2), 45.9 + 5.1 (Day 3), and 65.2 -t- 5.4 (Day 4) while control net IgE production (ng/mI) by
147
BRIEF COMMUNICATION mc
(A)
80
-
0
ol
;
' 0
I
I
I
I
I
0.01
0.1
1
5
10
JO
8-(B)
-8
a
-4
n X zi .-t 0
E > f:
*” @-+---+~-~-~-~ 2-
Ilk-9 AF-10
T
; 2 2 ;I 5 0
I I 1 1 1 0 0.01 0.1 1 5 10 Concentrations of NGF (rig/ml) FIG. 1. Effect of NGF on Ig production and cell growth by IM-9 and AF-10 cells. IM-9 (.A=) and AF-10 cells (OAU) were cultured with increasing concentrations of NGF for 4 days. IgG (0) and IgE production (0) in (A) and thymidine uptake (AA) and cell number (mEI) in (B) were determined. Vahtes are the means ? 1 SD of triplicate cultures. *Significant inhibition of Ig production (P < 0.01 compared to medium).
0
I
0
AF-10 was 3.2 + 0.6 (Day I), 17.0 + 0.7 (Day 2), 32.2 + 2.8 (Day 3), and 45.2 t 4.1 (Day 4). NGF did not inhibit Ig production by IM-9 or AF-10 when NGF was added to the culture after 1 day or when NGF was added to the culture at the initiation of culture then removed after l-3 days (data not shown). These results indicate that NGF must be added at the initiation of culture and must be present throughout the entire culture period. The specificity of the NGF effect was documented in Fig 2. NGF-induced inhibition of Ig production by IM-9 and AF-10 was blocked by anti-NGF serum but not by control serum. It has been reported that IL-3, IL-4, IL-S, and IL-6 affect plasma cell responses (6, 10, 11). However, IL-3 (0.1-10 U/ml), IL-4 (0.1-10 U/ml), IL-5 (0. I-10 &ml), and IL-6 (0. f-10 U/ml) did not affect Ig production or thymidine uptake by IM-9 or AF-IO (data not shown). In contrast, the addition of IL-6 restored the NGF-induced inhibition of Ig production while that of IL-3,
148
BRIEF COMMUNICATION IMP 9
t
AFmmIO
Medium
t-c
NGF NGF +anti-NGF NGF+ control
serum
NGF+ IL- 3 NGF+ IL-4 NGF+IL-
5
NGF +IL-
6 0
20
40
60
0
20
40
60
80
IgE Production (rig/ml) IgG Production (rig/ml) FIG. 2. Specificity of the NGF effect. IM-9 (W) and AF-IO cells (0) were cultured with the indicated factors. NGF was used at 5 &ml, sheep anti-NGF serum at I: 100, control sheep serum at 1: 100, IL-3 at 10 U/ml, IL-4 at 10 U/ml, IL-5 at IO &ml, and IL-6 at IO U/ml. Values are the means + I SD of triplicate cultures. *Significant inhibition of Ig production (P < 0.01 compared to medium).
IL-4, or IL-5 had no effect on the NGF-induced inhibition of Ig production (Fig. 2). The dose-response pattern of the IL-6 addition on NGF-induced Ig inhibition is shown in Table 1. As little as 1 U/ml of IL-6 significantly restored NGF-induced inhibition of Ig production, and inhibition was completely restored at a dose of 10 TABLE EFFECT
Factors Medium NGF NGF + IL-6 (0.1 U/ml) NGF + IL-6 (I U/ml) NGF + IL-6 (10 U/ml) NGF + IL-6 (IO U/ml) + anti-IL-6 antibody NGF + IL-6 (10 U/ml) + control antibody
OF IL-6
ON NGF-INDUCED
I INHIBITION
OF
Ig PRODUCTION
IM-9
AF-IO
IgG (rig/ml)
I@ (rig/ml)
56.6 23.2 24.8 39.4 55.8
(59)a,b (56)b (30)b (1)
45.3 18.1 19.5 30.4 44.6
(6O)b (57)b (33)b (2)
25.6 (55)b
19.0 (58)b
55.7 (2)
45.1 (0)
NOW. NGF was used at 5 r&ml, anti-IL-6 antibody at 2 &ml, and control antibody at 2 &ml. Values are the means of triplicate cultures. SD were
