Clin. exp. Immunol. (1991) 86, 537-543
Inter-mouse strain differences in the in vivo anti-CD3 induced cytokine release C. FERRAN*, M. DY*, K. SHEEHANt, S. MERITE*, R. SCHREIBERt, P. LANDAIS§, G. GRAU¶', J. BLUESTONE**, J.-F. BACH* & L. CHATENOUD* *INSERM U 25, CNRS UA 122, Hopital Necker, Paris, France, IDepartment of Pathology, Washington University School of Medicine, St Louis, MO, USA, §Laboratoire de biostatistique et d'informatique medicales, H6pital Necker, Paris, France, WHO Immunology Research and Training Center, Department of Pathology, University of Geneva, Geneva, Switzerland, and **Ben May Institute, University of Chicago, Chicago, Illinois, USA (Acceptedfor publication 7 June 1991)
SUMMARY
Triggering of the CD3 molecule by in vivo injection of the hamster anti-murine CD3 monoclonal antibody 145-2Cl in adult BALB/c mice leads to massive although transient T cell activation. High levels of tumour necrosis factor (TNF), interferon-gamma (IFN-y), IL-2, IL-3 and IL-6 are released into the circulation 1 to 8 h after a single 10 pg 145-2Cl i.v. injection. This release induces an impressive self-limited physical reaction associating hypothermia, hypomotility (as assessed by actimetry), diarrhoea, piloerection and even death when high doses (a single dose of > 100 pg/mouse injection) are administered. In vivo injection of 145-2C1 1 to other selected mouse strains, namely NZW, CBA/J and C3H/HeJ, induced both different cytokine release patterns and sickness. 145-2C1 induced significant release of TNF and IL-2 in all four strains. At variance, IFN-y was only detected in BALB/c mice sera which, in terms of physical reaction (hypothermia and hypomotility) were the most affected. Higher and long-lasting circulating IL-3/GM-CSF levels were present in CBA/J sera, correlating with a later recovery. These results underline heterogeneity in the in vivo cell activation pattern among different mouse strains, when triggering T lymphocytes via the CD3/Ti molecule as compared to exclusive targeting of monocyte/macrophages by means of lipopolysaccharide. Keywords T cell receptor CD3 monoclonal antibody serotherapy in vivo cell activation
kine release, although self-limited, is responsible in both humans and mice for a major acute clinical syndrome. The first OKT3 injection invariably induces fever, headache, chills, diarrhoea, and vomiting, and in a small proportion of patients (5-15%) more severe symptoms such as meningismus, respiratory distress and hypotension (Ortho Multicenter, 1985; Chatenoud et al., 1989). Similarly, as we already reported, injection of 145-2CI 1 into adult BALB/c mice (even a single injection of 10 pg i.v./mouse) induces severe hypothermia, somnolence, hypomotility, diarrhoea and piloerection. This reaction is lethal when doses higher than 100 pg/mouse are used (Ferran et al., 1990a, b). The physical reaction in mice is comparable to that seen when injecting lipopolysaccharide (LPS) or TNF. Interestingly, the intensity of the response to LPS varies considerably among inbred mouse strains, ranging from resistance to lethality (Sultzer, 1968; Glode et al., 1976; Watson, Largen & McAdam, 1978; Beutler et al., 1986a). The aim of this study was to compare the in vivo activating properties of 145-2CI I MoAb on four different mouse strains known to respond differently to LPS in terms of TNF produc-
INTRODUCTION The hamster monoclonal antibody (MoAb) 145-2C1 1 that specifically recognizes the E chain of the murine CD3 molecule (Leo et al., 1987) is a potent immunosuppressive agent able to significantly delay skin graft rejection in mice (Hirsch et al., 1988). In addition to its ability to suppress T cell function, in vivo administration of 145-2CI I induces potent although transient T cell activation. Shortly after 145-2C1 I treatment, high affinity IL-2 receptors are induced at the surface of spleen T cells (Hirsch et al., 1989) and massive release of some cytokines is observed (including exclusively T-cell-derived products) such as tumour necrosis factor (TNF), IL-2, IL-3, IL-6 and interferon-gamma (IFN-y) (Ferran et al., 1990a; Alegre et al., 1990). Similar immunosuppressive and T-cell-activation properties (i.e. cytokine release) are exhibited by an anti-human CD3 MoAb (OKT3) which is at present extensively used in clinical transplantation (Ortho Multicenter, 1985). Importantly, this cytoCorrespondence: Dr L. Chatenoud, INSERM U 25, HWpital Necker, 161 rue de Sevres, 75015 Paris, France.
537
538
C. Ferran et al.
tion: BALB/c, selected for their previously described reaction to anti-CD3 and good response to LPS; CBA/J, selected for their high production of TNF following LPS stimulation (Sultzer, 1968); NZW mice, for their recently described poor TNF production following LPS stimulation (Jacob & McDevitt, 1988); and C3H/HeJ mice, for their well-documented resistance to LPS stimulation (Glode et al., 1976).
MATERIALS AND METHODS Mice Female BALB/c, CBA/J, DBA/2 and Nude Swiss (nu/nu) mice 5 to 7 weeks old were purchased from Iffa Credo (L'Arbresle, France). C3H/HeJ were obtained from the Centre National de la Recherche Scientifique (CNRS) animal facilities (Orleans, France), and NZW mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All these animals were pathogen free. Monoclonal antibodies and treatment schedules 145-2C1 is an already described immunoglobulin G (IgG) hamster MoAb that is specific for the £ chain of the CD3 murine molecule. GK I .5 is a rat anti-mouse CD4 MoAb initially characterized by Dialynas et al. (1983). Monoclonal antibodies containing ascites were produced in sublethally irradiated (250 rad) DBA/2 mice for 145-2CI I and in nude mice for GK1.5. Ascites were purified by means of 500X) ammonium sulphate precipitation followed by ion exchange chromatography on DEAE columns. No endotoxin contamination (LAL assay) was detected in the 145-2C1 1 MoAb. Mice were injected i.v. with a single 10 pg dose of 145-2C II or GK1.5 diluted in saline (100 p1 total injected volume). The same dose of 145-2C1 1 was selected for in vivo injection of all four mice strains used. The choice of the dosage was based on both the criteria used for defining the dose in BALB/c mice as previously reported in detail (Ferran et al., 1990a, 1991) and on in vitro experiments analysing, in similar conventional culture conditions, the mitogenic response of splenocytes (obtained from the four mouse strains) to various concentrations of 1452C1 1. In all strains, similar 145-2C1 1 concentrations allowed for reaching the proliferation plateau (data not shown). In all actimetric studies, control mice injected with only 100 p1 of saline were studied in parallel with MoAb treated mice. Similar studies were also performed after a single intraperitoneal injection of 50 pg/mouse of an E. coli derived LPS (055 B5 E. coli LPS-Difco). Evaluation of motor activity The motor capacity of each mouse was measured by an actimetric device (Ferran et al., 1 990a). Motor activity of each mouse is quantified as the sum of the recorded photo-cell light interruptions in a predefined time interval. Measurements were made during the first 30 min after placing mice in the boxes of the Actimeter. Actimetry was performed before and at 4, 24 and 48 h following the injection of each tested MoAb. Both treated and control mice were marked to permit box changes at each time point to avoid habituation to the environment and facilitate longitudinal analysis of each animal. Six to eight animals were included in each studied group. Results represent the mean of four different experiments.
Body temperature and diarrhoea Mice were kept at room temperature of 22 to 24'C. Rectal body temperature was serially recorded before each actimetric study by means of an electronic heat sensitive microthermometer (Thermalet, TH8; Bailey Instruments). Diarrhoea was scored as present or absent at the same time points.
Cytokine assays Blood samples were drawn before and at 90 min and 4, 8 and 24 h following the MoAb injection. Blood was collected at 4"C in aseptic tubes devoid of additive and endotoxin-free (as assessed by the limulus assay). After clotting, sera were immediately recovered by centrifugation at 4-C and aliquots were stored at -80 C until tested for cytokine activities. Tumour necrosis factor. TNF was measured in parallel by means of a specific bioassay measuring the cytotoxic effect of sera on the L929 mouse fibroblast cell line, as described by Ferran et al. (1990a), and an ELISA, the technique of which has been reported in detail by Ferran et al. (1990a). Recombinant murine TNF (rMu TNF) was used to set up a standard curve for both techniques. rMu TNF produced in E. coli was purchased from Genentech (San Francisco, CA). Preparations displayed specific activity of 3 x I07 U/mg, and no endotoxin contamination was detected by the limulus assay. Interleukin-1. Proliferation of the human astrocytoma U373 cell line was used to test IL-1 activity according to the method detailed by Ferran et al. (1990a). This bioassay was selected because of its described specificity for IL-l. Recombinant purified murine IL-1 was used to set up the reference standard curve. rMu IL-I produced in E. coli was purchased from Roussel Uclaf (Romainville, France). Preparations displayed sp. act. of 5 x 107 U/mg, endotoxin contamination was less than 0-91 ng/mg of protein. Circulating IL-1 was assessed in our model in both mice sera and plasma (plasma was drawn in EDTA-supplemented tubes, LPS-free) after double chloroform extraction. To exclude the presence in the tested sera of any biological material exerting an inhibitory effect on the U373 proliferative capacity, recombinant IL- I was added to the tested sera. No inhibition of such a reference preparation could be evidenced. Interferon. Interferon was assessed by measuring inhibition of the cytopathic effect exerted by the vesicular stomatitis virus (VSV) on Res.L929 cells, a subline of L929 cells resistant to the cytotoxic effect of TNF. The test was performed as described in detail by Ferran et al. (1990a). Partially purified IFN-i/3 exhibiting a sp. act. of 107 U/mg and endotoxin-free as assessed by the limulus assay (kindly given by Dr I. Gresser, Villejuif, France) was used as a reference preparation. For inhibition experiments, sera were preincubated for 1 h at 37°C and 1 h at 4 C, at serial dilutions from 1/20 with the appropriate concentration of the hamster antimurine IFN-y MoAb H-22 before assessing the anti-viral activity. Interleukin-2. IL-2 activity was tested on the IL-2-dependent murine cytotoxic T cell line (CTLL-2) as described by Ferran et al. (1 990a). Reference curves were set up using both natural and recombinant IL-2, purchased from Roussel Uclaf, with a sp. act. of 107 U/mg and endotoxin-free as tested by the limulus assay. Inhibition tests were performed using two specific anti-murine IL-2 receptor MoAbs: 5A2 and PC61. For these experiments
539
Mouse strain differences in anti-CD3 induced cytokine release serial sera dilutions (starting at 1/30) were preincubated for 1 h at 37 C, then 1 h at 4 C, with either 5A2 or PC61 at the appropriate concentration, before assessing the proliferative capacity on CTLL-2. Interleukin-3/GM-CSF. Histamine cell producing stimulating activity (HCSA) was the assay used for measuring IL-3/GMCSF (Ferran et al., 1990a) because of the described inhibitory effect of murine sera on the classical IL-3 bioassays. This activity was shown to be mediated by both IL-3 and GM-CSF. In our hands 145-2C1 1 exhibited minor HCSA. Thus, HCSA in the sera of 145-2C1 1 treated mice were corrected for this background value. Inhibition tests were performed by preincubating 3 h at 37 C serum samples with specific goat anti-murine IL-3 and/or rabbit anti-murine GM-CSF polyclonal antibodies (anti-murine IL-3 antibodies were a kind gift of Dr H. Ziltener, Vancouver, Canada, and anti-murine GM-CSF antibodies were a kind gift of Dr Mermod, BIOGEN, Geneva, Switzerland). Statistical analysis Mouse motor activity with time, according to the treatment administered, was analysed by analysis of variance (ANOVA) and repeated measurements. Post hoc tests were done to delineate temporal differences between treatment and placebo at each time. The analysis was made using NCSS software. All the other statistical analyses were performed using Student's t-test.
RESULTS Physical reaction to 145-2CJJ treatment Animals were injected with a single dose of 10 pg 145-2C1 1 i.v./ mouse. In preliminary experiments (results not shown), to define the maximal reaction and total recovery times, kinetic studies were performed at 1, 2, 4, 5, 7, 8 and 24 h after one injection of 1452CI 1 at 10 pg/mouse. These studies led us to select 4, 24, 48 and 72 h after anti-CD3 injection for measuring body temperature and motility. Actimetric evaluation at each time point reflected the overall receptive and reactive capacity of the animals. Hypomotility was consistently observed after 145-2C 1I treatment. Body temperature was also highly modified since high levels of circulating TNF and IL- 1 lead generally to hypothermia (Ferran et al., 1990a). All experiments showed very good reproducibility. Treated and control groups included six to eight animals each. Actimetric evaluation. (See Fig. 1.) Before anti-CD3 injection, mean activity (+ s.e.m.) of normal mice varied among strains: 372 + 19 movements per 30 min (mvts/30') in BALB/c mice 359 + 27 mvts/30' in NZW mice 211 + 19 mvts/30' in CBA/J mice 230 + 15 mvts/30' in C3H/HeJ mice
BALB/c mice presented a significant decrease in motor capacity (16% of control) 4 h following 145-2Cl 1 injection (P < 0-0001) (Fig. 1). At this same time point NZW, CBA/J and C3H/HeJ showed moderate hypomotility ranging 43%, 63% and 60% of controls (P < 0-001) (Fig. 1). For all four strains, this hypomotility reversed partially by 24 h, and at 48 h, BALB/c, NZW and C3H/HeJ had completely recovered (Fig. 1). Conversely, CBA/J mice still presented significant hypomotility at
I BALB/c
0 0
0A
CBA/J
C3H/HeJ
500
50
0
4
24
48
0 72 Time ( h)
4
24
48
72
Fig. 1. Actimetric evaluation of BALB/c, NZW, CBA/J and C3H/HeJ mice after one single 10 pg injection of 145-2C1 1 (1) compared at each time with controls treated with saline (0). Results are expressed in % motility of controls at each time point. Six to eight animals were included in each group. Results represent the mean of six different experiments; number of movements/30 min. ***P
Inter-mouse strain differences in the in vivo anti-CD3 induced cytokine release C. FERRAN*, M. DY*, K. SHEEHANt, S. MERITE*, R. SCHREIBERt, P. LANDAIS§, G. GRAU¶', J. BLUESTONE**, J.-F. BACH* & L. CHATENOUD* *INSERM U 25, CNRS UA 122, Hopital Necker, Paris, France, IDepartment of Pathology, Washington University School of Medicine, St Louis, MO, USA, §Laboratoire de biostatistique et d'informatique medicales, H6pital Necker, Paris, France, WHO Immunology Research and Training Center, Department of Pathology, University of Geneva, Geneva, Switzerland, and **Ben May Institute, University of Chicago, Chicago, Illinois, USA (Acceptedfor publication 7 June 1991)
SUMMARY
Triggering of the CD3 molecule by in vivo injection of the hamster anti-murine CD3 monoclonal antibody 145-2Cl in adult BALB/c mice leads to massive although transient T cell activation. High levels of tumour necrosis factor (TNF), interferon-gamma (IFN-y), IL-2, IL-3 and IL-6 are released into the circulation 1 to 8 h after a single 10 pg 145-2Cl i.v. injection. This release induces an impressive self-limited physical reaction associating hypothermia, hypomotility (as assessed by actimetry), diarrhoea, piloerection and even death when high doses (a single dose of > 100 pg/mouse injection) are administered. In vivo injection of 145-2C1 1 to other selected mouse strains, namely NZW, CBA/J and C3H/HeJ, induced both different cytokine release patterns and sickness. 145-2C1 induced significant release of TNF and IL-2 in all four strains. At variance, IFN-y was only detected in BALB/c mice sera which, in terms of physical reaction (hypothermia and hypomotility) were the most affected. Higher and long-lasting circulating IL-3/GM-CSF levels were present in CBA/J sera, correlating with a later recovery. These results underline heterogeneity in the in vivo cell activation pattern among different mouse strains, when triggering T lymphocytes via the CD3/Ti molecule as compared to exclusive targeting of monocyte/macrophages by means of lipopolysaccharide. Keywords T cell receptor CD3 monoclonal antibody serotherapy in vivo cell activation
kine release, although self-limited, is responsible in both humans and mice for a major acute clinical syndrome. The first OKT3 injection invariably induces fever, headache, chills, diarrhoea, and vomiting, and in a small proportion of patients (5-15%) more severe symptoms such as meningismus, respiratory distress and hypotension (Ortho Multicenter, 1985; Chatenoud et al., 1989). Similarly, as we already reported, injection of 145-2CI 1 into adult BALB/c mice (even a single injection of 10 pg i.v./mouse) induces severe hypothermia, somnolence, hypomotility, diarrhoea and piloerection. This reaction is lethal when doses higher than 100 pg/mouse are used (Ferran et al., 1990a, b). The physical reaction in mice is comparable to that seen when injecting lipopolysaccharide (LPS) or TNF. Interestingly, the intensity of the response to LPS varies considerably among inbred mouse strains, ranging from resistance to lethality (Sultzer, 1968; Glode et al., 1976; Watson, Largen & McAdam, 1978; Beutler et al., 1986a). The aim of this study was to compare the in vivo activating properties of 145-2CI I MoAb on four different mouse strains known to respond differently to LPS in terms of TNF produc-
INTRODUCTION The hamster monoclonal antibody (MoAb) 145-2C1 1 that specifically recognizes the E chain of the murine CD3 molecule (Leo et al., 1987) is a potent immunosuppressive agent able to significantly delay skin graft rejection in mice (Hirsch et al., 1988). In addition to its ability to suppress T cell function, in vivo administration of 145-2CI I induces potent although transient T cell activation. Shortly after 145-2C1 I treatment, high affinity IL-2 receptors are induced at the surface of spleen T cells (Hirsch et al., 1989) and massive release of some cytokines is observed (including exclusively T-cell-derived products) such as tumour necrosis factor (TNF), IL-2, IL-3, IL-6 and interferon-gamma (IFN-y) (Ferran et al., 1990a; Alegre et al., 1990). Similar immunosuppressive and T-cell-activation properties (i.e. cytokine release) are exhibited by an anti-human CD3 MoAb (OKT3) which is at present extensively used in clinical transplantation (Ortho Multicenter, 1985). Importantly, this cytoCorrespondence: Dr L. Chatenoud, INSERM U 25, HWpital Necker, 161 rue de Sevres, 75015 Paris, France.
537
538
C. Ferran et al.
tion: BALB/c, selected for their previously described reaction to anti-CD3 and good response to LPS; CBA/J, selected for their high production of TNF following LPS stimulation (Sultzer, 1968); NZW mice, for their recently described poor TNF production following LPS stimulation (Jacob & McDevitt, 1988); and C3H/HeJ mice, for their well-documented resistance to LPS stimulation (Glode et al., 1976).
MATERIALS AND METHODS Mice Female BALB/c, CBA/J, DBA/2 and Nude Swiss (nu/nu) mice 5 to 7 weeks old were purchased from Iffa Credo (L'Arbresle, France). C3H/HeJ were obtained from the Centre National de la Recherche Scientifique (CNRS) animal facilities (Orleans, France), and NZW mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All these animals were pathogen free. Monoclonal antibodies and treatment schedules 145-2C1 is an already described immunoglobulin G (IgG) hamster MoAb that is specific for the £ chain of the CD3 murine molecule. GK I .5 is a rat anti-mouse CD4 MoAb initially characterized by Dialynas et al. (1983). Monoclonal antibodies containing ascites were produced in sublethally irradiated (250 rad) DBA/2 mice for 145-2CI I and in nude mice for GK1.5. Ascites were purified by means of 500X) ammonium sulphate precipitation followed by ion exchange chromatography on DEAE columns. No endotoxin contamination (LAL assay) was detected in the 145-2C1 1 MoAb. Mice were injected i.v. with a single 10 pg dose of 145-2C II or GK1.5 diluted in saline (100 p1 total injected volume). The same dose of 145-2C1 1 was selected for in vivo injection of all four mice strains used. The choice of the dosage was based on both the criteria used for defining the dose in BALB/c mice as previously reported in detail (Ferran et al., 1990a, 1991) and on in vitro experiments analysing, in similar conventional culture conditions, the mitogenic response of splenocytes (obtained from the four mouse strains) to various concentrations of 1452C1 1. In all strains, similar 145-2C1 1 concentrations allowed for reaching the proliferation plateau (data not shown). In all actimetric studies, control mice injected with only 100 p1 of saline were studied in parallel with MoAb treated mice. Similar studies were also performed after a single intraperitoneal injection of 50 pg/mouse of an E. coli derived LPS (055 B5 E. coli LPS-Difco). Evaluation of motor activity The motor capacity of each mouse was measured by an actimetric device (Ferran et al., 1 990a). Motor activity of each mouse is quantified as the sum of the recorded photo-cell light interruptions in a predefined time interval. Measurements were made during the first 30 min after placing mice in the boxes of the Actimeter. Actimetry was performed before and at 4, 24 and 48 h following the injection of each tested MoAb. Both treated and control mice were marked to permit box changes at each time point to avoid habituation to the environment and facilitate longitudinal analysis of each animal. Six to eight animals were included in each studied group. Results represent the mean of four different experiments.
Body temperature and diarrhoea Mice were kept at room temperature of 22 to 24'C. Rectal body temperature was serially recorded before each actimetric study by means of an electronic heat sensitive microthermometer (Thermalet, TH8; Bailey Instruments). Diarrhoea was scored as present or absent at the same time points.
Cytokine assays Blood samples were drawn before and at 90 min and 4, 8 and 24 h following the MoAb injection. Blood was collected at 4"C in aseptic tubes devoid of additive and endotoxin-free (as assessed by the limulus assay). After clotting, sera were immediately recovered by centrifugation at 4-C and aliquots were stored at -80 C until tested for cytokine activities. Tumour necrosis factor. TNF was measured in parallel by means of a specific bioassay measuring the cytotoxic effect of sera on the L929 mouse fibroblast cell line, as described by Ferran et al. (1990a), and an ELISA, the technique of which has been reported in detail by Ferran et al. (1990a). Recombinant murine TNF (rMu TNF) was used to set up a standard curve for both techniques. rMu TNF produced in E. coli was purchased from Genentech (San Francisco, CA). Preparations displayed specific activity of 3 x I07 U/mg, and no endotoxin contamination was detected by the limulus assay. Interleukin-1. Proliferation of the human astrocytoma U373 cell line was used to test IL-1 activity according to the method detailed by Ferran et al. (1990a). This bioassay was selected because of its described specificity for IL-l. Recombinant purified murine IL-1 was used to set up the reference standard curve. rMu IL-I produced in E. coli was purchased from Roussel Uclaf (Romainville, France). Preparations displayed sp. act. of 5 x 107 U/mg, endotoxin contamination was less than 0-91 ng/mg of protein. Circulating IL-1 was assessed in our model in both mice sera and plasma (plasma was drawn in EDTA-supplemented tubes, LPS-free) after double chloroform extraction. To exclude the presence in the tested sera of any biological material exerting an inhibitory effect on the U373 proliferative capacity, recombinant IL- I was added to the tested sera. No inhibition of such a reference preparation could be evidenced. Interferon. Interferon was assessed by measuring inhibition of the cytopathic effect exerted by the vesicular stomatitis virus (VSV) on Res.L929 cells, a subline of L929 cells resistant to the cytotoxic effect of TNF. The test was performed as described in detail by Ferran et al. (1990a). Partially purified IFN-i/3 exhibiting a sp. act. of 107 U/mg and endotoxin-free as assessed by the limulus assay (kindly given by Dr I. Gresser, Villejuif, France) was used as a reference preparation. For inhibition experiments, sera were preincubated for 1 h at 37°C and 1 h at 4 C, at serial dilutions from 1/20 with the appropriate concentration of the hamster antimurine IFN-y MoAb H-22 before assessing the anti-viral activity. Interleukin-2. IL-2 activity was tested on the IL-2-dependent murine cytotoxic T cell line (CTLL-2) as described by Ferran et al. (1 990a). Reference curves were set up using both natural and recombinant IL-2, purchased from Roussel Uclaf, with a sp. act. of 107 U/mg and endotoxin-free as tested by the limulus assay. Inhibition tests were performed using two specific anti-murine IL-2 receptor MoAbs: 5A2 and PC61. For these experiments
539
Mouse strain differences in anti-CD3 induced cytokine release serial sera dilutions (starting at 1/30) were preincubated for 1 h at 37 C, then 1 h at 4 C, with either 5A2 or PC61 at the appropriate concentration, before assessing the proliferative capacity on CTLL-2. Interleukin-3/GM-CSF. Histamine cell producing stimulating activity (HCSA) was the assay used for measuring IL-3/GMCSF (Ferran et al., 1990a) because of the described inhibitory effect of murine sera on the classical IL-3 bioassays. This activity was shown to be mediated by both IL-3 and GM-CSF. In our hands 145-2C1 1 exhibited minor HCSA. Thus, HCSA in the sera of 145-2C1 1 treated mice were corrected for this background value. Inhibition tests were performed by preincubating 3 h at 37 C serum samples with specific goat anti-murine IL-3 and/or rabbit anti-murine GM-CSF polyclonal antibodies (anti-murine IL-3 antibodies were a kind gift of Dr H. Ziltener, Vancouver, Canada, and anti-murine GM-CSF antibodies were a kind gift of Dr Mermod, BIOGEN, Geneva, Switzerland). Statistical analysis Mouse motor activity with time, according to the treatment administered, was analysed by analysis of variance (ANOVA) and repeated measurements. Post hoc tests were done to delineate temporal differences between treatment and placebo at each time. The analysis was made using NCSS software. All the other statistical analyses were performed using Student's t-test.
RESULTS Physical reaction to 145-2CJJ treatment Animals were injected with a single dose of 10 pg 145-2C1 1 i.v./ mouse. In preliminary experiments (results not shown), to define the maximal reaction and total recovery times, kinetic studies were performed at 1, 2, 4, 5, 7, 8 and 24 h after one injection of 1452CI 1 at 10 pg/mouse. These studies led us to select 4, 24, 48 and 72 h after anti-CD3 injection for measuring body temperature and motility. Actimetric evaluation at each time point reflected the overall receptive and reactive capacity of the animals. Hypomotility was consistently observed after 145-2C 1I treatment. Body temperature was also highly modified since high levels of circulating TNF and IL- 1 lead generally to hypothermia (Ferran et al., 1990a). All experiments showed very good reproducibility. Treated and control groups included six to eight animals each. Actimetric evaluation. (See Fig. 1.) Before anti-CD3 injection, mean activity (+ s.e.m.) of normal mice varied among strains: 372 + 19 movements per 30 min (mvts/30') in BALB/c mice 359 + 27 mvts/30' in NZW mice 211 + 19 mvts/30' in CBA/J mice 230 + 15 mvts/30' in C3H/HeJ mice
BALB/c mice presented a significant decrease in motor capacity (16% of control) 4 h following 145-2Cl 1 injection (P < 0-0001) (Fig. 1). At this same time point NZW, CBA/J and C3H/HeJ showed moderate hypomotility ranging 43%, 63% and 60% of controls (P < 0-001) (Fig. 1). For all four strains, this hypomotility reversed partially by 24 h, and at 48 h, BALB/c, NZW and C3H/HeJ had completely recovered (Fig. 1). Conversely, CBA/J mice still presented significant hypomotility at
I BALB/c
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C3H/HeJ
500
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0 72 Time ( h)
4
24
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Fig. 1. Actimetric evaluation of BALB/c, NZW, CBA/J and C3H/HeJ mice after one single 10 pg injection of 145-2C1 1 (1) compared at each time with controls treated with saline (0). Results are expressed in % motility of controls at each time point. Six to eight animals were included in each group. Results represent the mean of six different experiments; number of movements/30 min. ***P
