Veterinarylmmunology andlmmunopathology, 28 ( 1991 ) 157-163 Elsevier Science Publishers B.V., Amsterdam
157
Pasteurella haemolytica A I purified capsular polysaccharide does not stimulate interleukin-1 and tumor necrosis factor release by bovine monocytes and alveolar macrophages Charles J. Czuprynski a, E.J. Noel a and C. Adlam b aDepartment of Pathobiological Sciences, School of VeterinaryMedicine, Universityof Wisconsin, Madison, W153706, USA bDepartment of Bacteriology, The WeilcomeResearch Laboratories, Beckenham, Kent, UK (Accepted 8 June 1990)
ABSTRACT Czuprynski, C.J., Noel, E.J. and Adlam, C., 1991. Pasteurella haemolytica A l purified capsular polysaccharide does not stimulate interleukin-l and tumor necrosis factor release by bovine monocytes and alveolar macrophages. Vet.Immunol. Immunopathol., 28:157-163. Purified capsular polysaccharide (CPS) stimulated significant release of interleukin-1 (IL- 1) activity from bovine blood monocytes but not alveolar macrophages in vitro. The ability of CPS to induce IL-l release was resistant to boiling and inhibited by the addition of polymyxinft. Thus, it is likelv that the IL-1 release stimulated by CPR regultad frnm t h e c m a l l amn,,nt of contaminating li,,,~ polysaccharide (LPS) that was present (an estimated 5 pg LPS per pg CPS) rather than to a direct effect of CPS. Tumor necrosis factor activity was not detected in the culture supernatants of bovine monocytes incubated with purified CPS for 1-18 h in vitro.
INTRODUCTION
Pasteurella haemolytica A 1 causes an acute p l e u r o p n e u m o n i a in cattle that is o f considerable e c o n o m i c i m p o r t a n c e ( C o n f e r et al., 1988). At least two virulence d e t e r m i n a n t s have been p r o p o s e d to c o n t r i b u t e to the pathogenesis o f p u l m o n a r y pasteurellosis. T h e first is a p o t e n t cytotoxin that directly impairs and eventually kills phagocytic cells (Sh~,'~,¢a a n d W;lkie, ~o87 ), T h e second is a loosely attached c a p s u l a r polysaccharide (Beesley et al., 1982; G e n t r y et al., 1982; G i l m o u r et al., 1985; M o r c k et al., 1987) whose chemical structure has been defined as -3 ) - M a n N A e - ( f l l - 4 ) - M a n N A c A - ( B l ( A d l a m et al., 1984). P r e v i o u s reports h a v e indicated that P. haemolytica A l capsular polysaccharide is deposited in the lungs of cattle with p u l m o n a r y pasteurellosis (Whitley et el., 1989), a n d that capsular polysaccharide inhibits the
0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
158
C.J. CZUPRYNSKI ET AL.
ingestion and killing of P. haemolytica by bovine neutrophils (Czuprynski et al., 1989) and alveolar macrophages in vitro (Czuprynski, et al., 1991 ). It is not known whether this capsular polysaccharide stimulates the release of inflammatory mediators that contribute to the fibrinous pleuropneumonia that characterizes pulmonary pasteurellosis. Interleukin-1 (IL-1 ) and tumor necrosis factor (TNF) are two potent inflammatory mediators that are released by mononuclear phagocytes stimulated with bacterial products (Le and Vilcek, 1987). In the present study we examined the effects ofP. haemolytica A 1 purified capsular polysaccharide (CPS) on the release of IL- 1 and TNF by bovine monocytes and alveolar macrophages in vitro. MATERIALS A N D M E T H O D S
Media and reagents RPMI-1640, DMEM, and fetal bovine serum (FBS) (lot # 29N9063) were purchased from GIBCO Laboratories (Madison, WI). Purified phytohaemaglutinin A (PHA) was obtained from Burroughs Wellcome Co., (Greenville, NC). Escherichia coli 055 :B5 lipopolysaccharide (LPS-W) was purchased from Difco Laboratories (Detroit, MI ). P. haemolytica A 1 purified CPS CPS was purified from_the culture supernatant of a broth culture of P. haemolytica AI as described previously in detail (Adlam et al., 1984). We have demonstrated previously that CPS is not cytotoxic for bovine leukocytes (Czuprynski et al., 1989). It contains approximately 5 pg of LPS per gg CPS, as determined by the iimuius amoebocyte lysate assay (Sigma, St. Louis, MO ).
Preparation of alveolar macrophages and blood monocytes Lung cells were recovered, as descr/bed previously (Lederer et al., 1987), by twice lavaging the excised lungs of freshly killed cattle with sterile pHbalanced (7.2) saline that contained 0.2% EDTA. The lung cells were washed three times (300 × g for 10 min at 4 ° C) and suspended at 4.1 X 106 viable cells per ml in Hanks' balanced salts solution with 0.25 bovine serum albumin (HBSA). Cell viability was measured by trypan blue exclusion (average viability of 85%). Lung cell suspensions contained an average of 92% alveolar macrophages as determined by microscopic examination of Diff-Quik-stained cytospin smears. To obtain peripheral blood monocytes, citrated blood was centrifuged (300×g for 20 min at 22°C), the buffy coat cells were removed and centrifuged on Ficoll-Hypaque gradients to obtain mononuclear cell-rich populations. Bovine blood monocytes were obtained by allowing the mononuclear cells to adhere to plastic tissue culture wells for 2 h at 39°C. After removal of the nonadherent cells the adherent monolayer was determined to be 85-95%
EFFECT OF P. HAEMOLYTICA CAPSULAR POLYSACCHARIDE ON MONOCYTES/MACROPHAGES
159
monocytes and greater than 95% viable as described previously (Zurbrick et al., 1986).
Production and detection of lL-1 and TNF activity Peripheral blood monocytes and alveolar macrophages (2 × 106 per well) were incubated for 18-20 h at 39°C in RPMI containing 10% FBS in 24-well tissue culture plates (Falcom, Oxnard, CA). Some wells received 10/tg LPS or 0.1 to 10/tg CPS. IL-l activity was assessed by the murine C3H/HeJ thymocyte assay as described previously (Lederer and Czuprynski, 1989). Briefly, thymocytes ( 7 X 105 per well ), PHA (0.4/zg ), and various dilutions of culture supernatants (0.05 ml per well) were added in triplicate to flat-bottomed 96well tissue culture plates (Costar, Cambridge, MA) and incubated for 72 h at 37°C in 5% CO2. The cells were pulsed with [3H]-thymidine for the last 6 h of culture and radiolabel incorporation was determined by liquid scintillation spectrophotometry. TNF activity was determined using WEHI- 164 cells in a cytotoxicity assay as described previously (Adams et al., 1990). Briefly, various dilutions of monocyte culture supernatants were incubated with actinomycin D treated WEHI-164 cells for 18 h at 39°C. Following this, the cells were fixed and stained with crystal violet. The stain was then solubilized and the optical density determined using an automated microELISA plate reader. RESULTS
Bovine blood monocytes (Fig. I A) released significant amounts of IL-1 activity when stimulated with l 0 gg CPS (P < 0.05 ). The amount of IL- l activity released, however, was significantly less (P < 0.05 ) than that stimulated by the addition of l 0 gg LPS (Fig. l A). In contrast, bovine alveolar macrophages failed to release IL- l in response to CPS and released less IL- l activity in response to LPS then did bovine monocytes (Fig. l B). Bovine monocytes did not release detectable TNF activity when incubated with l 0 gg CPS for l - 18 h in vitro (data not shown ). We were concerned that IL- 1 release from monocytes stimulated with CPS might have been confounded by the presence of contaminating LPS. We, therefore, tested the effects of heat treatment (boiling for I h) and polymyxin fl on the ability of CPS to stimulate IL-l release. Our results indicated that the ability of CPS to induce !L-! release was largely ~esistant to boiling, and that the addition of polymyxin fl significantly reduced (P < 0.05 ), but did not altogether eliminate, the release of IL-1 from monocytes (Table 1 ). Both of these observations were similar to what we observed with monocytes stimulated with an amount of exogenous LPS (5 pg) equivalent to that estimated to be present in 1 gg CPS.
160
C.J. CZUPRYNSKI ET AL.
A
MONOC'Iq'ES
v,X Q.
o
I
I
441
ALVEOLARMACROPHAGES
Z
o
8 z
i
20
'° .
UNSTIM
LPS
CPS
10,~j/ml 10~j/mi
CPS
.
CPS
1~j/rni 0.1/~g/rnl
Fig. 1. Purified CPS stimulates the release of IL-1 activity from bovine monocytes but not from alveolar macrophages. Unstimulated and LPS-stimulated monocytes and macrophages were included as negative and positive controls, respectively. Cells were cultured with the indicated stimuli for 18-24 h and the culture supernatants were removed and assayed for IL-I activity using the C 3 H / H e J thymocyte co-mitogenesis assay. The results indicate the mean + SEM of four (macrophages) and six (monocytes) separate experiments. TABLE 1 Capsular polysaccharide-stimulated release ofinterleukin- 1 activity by bovine monocytes may be due to contaminating LPS Monocyte treatmenP
Mean cpm + SEM ( × 10 3 )
CPS CPS + polymyxin p CPS boiled LPS LPS+ polymyxin fl LPS boiled Unstimulated Unstimulated + polymyxin fl Media control
17.1 +6.8 11.2 + 0.9 b 23.4 + 6.1 30.6 + 6.5 12.5 + 1.4c 28.6 + 6.6 23.2 + 5.7 11.2 + 0.4 d 3.0 + 1.1
[ 3H ]-thymidine incorporation was determined and compared to thymocytes stimulated with PHA alone (3.0 + I. I cpm × 103). The results are the mean + SEM of three separate experiments. aBovine adherent monocyte were cultured as indicated for 18-20 h with 1/zg per ml, 5 pg per ml LPS or 1/zg per ml polymyxin p. IL-1 activity was detected by adding culture supernatants to C3H/HeJ mouse thymocytes stimulated with a suboptimal concentration of PHA. bp< 0.05 compared to monocytes treated with CPS. , ~ _ ,~ n¢ C~,~..p~r~rl t¢~ m c m o c v t e s t r e a t e d w i t h L P S . dp
157
Pasteurella haemolytica A I purified capsular polysaccharide does not stimulate interleukin-1 and tumor necrosis factor release by bovine monocytes and alveolar macrophages Charles J. Czuprynski a, E.J. Noel a and C. Adlam b aDepartment of Pathobiological Sciences, School of VeterinaryMedicine, Universityof Wisconsin, Madison, W153706, USA bDepartment of Bacteriology, The WeilcomeResearch Laboratories, Beckenham, Kent, UK (Accepted 8 June 1990)
ABSTRACT Czuprynski, C.J., Noel, E.J. and Adlam, C., 1991. Pasteurella haemolytica A l purified capsular polysaccharide does not stimulate interleukin-l and tumor necrosis factor release by bovine monocytes and alveolar macrophages. Vet.Immunol. Immunopathol., 28:157-163. Purified capsular polysaccharide (CPS) stimulated significant release of interleukin-1 (IL- 1) activity from bovine blood monocytes but not alveolar macrophages in vitro. The ability of CPS to induce IL-l release was resistant to boiling and inhibited by the addition of polymyxinft. Thus, it is likelv that the IL-1 release stimulated by CPR regultad frnm t h e c m a l l amn,,nt of contaminating li,,,~ polysaccharide (LPS) that was present (an estimated 5 pg LPS per pg CPS) rather than to a direct effect of CPS. Tumor necrosis factor activity was not detected in the culture supernatants of bovine monocytes incubated with purified CPS for 1-18 h in vitro.
INTRODUCTION
Pasteurella haemolytica A 1 causes an acute p l e u r o p n e u m o n i a in cattle that is o f considerable e c o n o m i c i m p o r t a n c e ( C o n f e r et al., 1988). At least two virulence d e t e r m i n a n t s have been p r o p o s e d to c o n t r i b u t e to the pathogenesis o f p u l m o n a r y pasteurellosis. T h e first is a p o t e n t cytotoxin that directly impairs and eventually kills phagocytic cells (Sh~,'~,¢a a n d W;lkie, ~o87 ), T h e second is a loosely attached c a p s u l a r polysaccharide (Beesley et al., 1982; G e n t r y et al., 1982; G i l m o u r et al., 1985; M o r c k et al., 1987) whose chemical structure has been defined as -3 ) - M a n N A e - ( f l l - 4 ) - M a n N A c A - ( B l ( A d l a m et al., 1984). P r e v i o u s reports h a v e indicated that P. haemolytica A l capsular polysaccharide is deposited in the lungs of cattle with p u l m o n a r y pasteurellosis (Whitley et el., 1989), a n d that capsular polysaccharide inhibits the
0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
158
C.J. CZUPRYNSKI ET AL.
ingestion and killing of P. haemolytica by bovine neutrophils (Czuprynski et al., 1989) and alveolar macrophages in vitro (Czuprynski, et al., 1991 ). It is not known whether this capsular polysaccharide stimulates the release of inflammatory mediators that contribute to the fibrinous pleuropneumonia that characterizes pulmonary pasteurellosis. Interleukin-1 (IL-1 ) and tumor necrosis factor (TNF) are two potent inflammatory mediators that are released by mononuclear phagocytes stimulated with bacterial products (Le and Vilcek, 1987). In the present study we examined the effects ofP. haemolytica A 1 purified capsular polysaccharide (CPS) on the release of IL- 1 and TNF by bovine monocytes and alveolar macrophages in vitro. MATERIALS A N D M E T H O D S
Media and reagents RPMI-1640, DMEM, and fetal bovine serum (FBS) (lot # 29N9063) were purchased from GIBCO Laboratories (Madison, WI). Purified phytohaemaglutinin A (PHA) was obtained from Burroughs Wellcome Co., (Greenville, NC). Escherichia coli 055 :B5 lipopolysaccharide (LPS-W) was purchased from Difco Laboratories (Detroit, MI ). P. haemolytica A 1 purified CPS CPS was purified from_the culture supernatant of a broth culture of P. haemolytica AI as described previously in detail (Adlam et al., 1984). We have demonstrated previously that CPS is not cytotoxic for bovine leukocytes (Czuprynski et al., 1989). It contains approximately 5 pg of LPS per gg CPS, as determined by the iimuius amoebocyte lysate assay (Sigma, St. Louis, MO ).
Preparation of alveolar macrophages and blood monocytes Lung cells were recovered, as descr/bed previously (Lederer et al., 1987), by twice lavaging the excised lungs of freshly killed cattle with sterile pHbalanced (7.2) saline that contained 0.2% EDTA. The lung cells were washed three times (300 × g for 10 min at 4 ° C) and suspended at 4.1 X 106 viable cells per ml in Hanks' balanced salts solution with 0.25 bovine serum albumin (HBSA). Cell viability was measured by trypan blue exclusion (average viability of 85%). Lung cell suspensions contained an average of 92% alveolar macrophages as determined by microscopic examination of Diff-Quik-stained cytospin smears. To obtain peripheral blood monocytes, citrated blood was centrifuged (300×g for 20 min at 22°C), the buffy coat cells were removed and centrifuged on Ficoll-Hypaque gradients to obtain mononuclear cell-rich populations. Bovine blood monocytes were obtained by allowing the mononuclear cells to adhere to plastic tissue culture wells for 2 h at 39°C. After removal of the nonadherent cells the adherent monolayer was determined to be 85-95%
EFFECT OF P. HAEMOLYTICA CAPSULAR POLYSACCHARIDE ON MONOCYTES/MACROPHAGES
159
monocytes and greater than 95% viable as described previously (Zurbrick et al., 1986).
Production and detection of lL-1 and TNF activity Peripheral blood monocytes and alveolar macrophages (2 × 106 per well) were incubated for 18-20 h at 39°C in RPMI containing 10% FBS in 24-well tissue culture plates (Falcom, Oxnard, CA). Some wells received 10/tg LPS or 0.1 to 10/tg CPS. IL-l activity was assessed by the murine C3H/HeJ thymocyte assay as described previously (Lederer and Czuprynski, 1989). Briefly, thymocytes ( 7 X 105 per well ), PHA (0.4/zg ), and various dilutions of culture supernatants (0.05 ml per well) were added in triplicate to flat-bottomed 96well tissue culture plates (Costar, Cambridge, MA) and incubated for 72 h at 37°C in 5% CO2. The cells were pulsed with [3H]-thymidine for the last 6 h of culture and radiolabel incorporation was determined by liquid scintillation spectrophotometry. TNF activity was determined using WEHI- 164 cells in a cytotoxicity assay as described previously (Adams et al., 1990). Briefly, various dilutions of monocyte culture supernatants were incubated with actinomycin D treated WEHI-164 cells for 18 h at 39°C. Following this, the cells were fixed and stained with crystal violet. The stain was then solubilized and the optical density determined using an automated microELISA plate reader. RESULTS
Bovine blood monocytes (Fig. I A) released significant amounts of IL-1 activity when stimulated with l 0 gg CPS (P < 0.05 ). The amount of IL- l activity released, however, was significantly less (P < 0.05 ) than that stimulated by the addition of l 0 gg LPS (Fig. l A). In contrast, bovine alveolar macrophages failed to release IL- l in response to CPS and released less IL- l activity in response to LPS then did bovine monocytes (Fig. l B). Bovine monocytes did not release detectable TNF activity when incubated with l 0 gg CPS for l - 18 h in vitro (data not shown ). We were concerned that IL- 1 release from monocytes stimulated with CPS might have been confounded by the presence of contaminating LPS. We, therefore, tested the effects of heat treatment (boiling for I h) and polymyxin fl on the ability of CPS to stimulate IL-l release. Our results indicated that the ability of CPS to induce !L-! release was largely ~esistant to boiling, and that the addition of polymyxin fl significantly reduced (P < 0.05 ), but did not altogether eliminate, the release of IL-1 from monocytes (Table 1 ). Both of these observations were similar to what we observed with monocytes stimulated with an amount of exogenous LPS (5 pg) equivalent to that estimated to be present in 1 gg CPS.
160
C.J. CZUPRYNSKI ET AL.
A
MONOC'Iq'ES
v,X Q.
o
I
I
441
ALVEOLARMACROPHAGES
Z
o
8 z
i
20
'° .
UNSTIM
LPS
CPS
10,~j/ml 10~j/mi
CPS
.
CPS
1~j/rni 0.1/~g/rnl
Fig. 1. Purified CPS stimulates the release of IL-1 activity from bovine monocytes but not from alveolar macrophages. Unstimulated and LPS-stimulated monocytes and macrophages were included as negative and positive controls, respectively. Cells were cultured with the indicated stimuli for 18-24 h and the culture supernatants were removed and assayed for IL-I activity using the C 3 H / H e J thymocyte co-mitogenesis assay. The results indicate the mean + SEM of four (macrophages) and six (monocytes) separate experiments. TABLE 1 Capsular polysaccharide-stimulated release ofinterleukin- 1 activity by bovine monocytes may be due to contaminating LPS Monocyte treatmenP
Mean cpm + SEM ( × 10 3 )
CPS CPS + polymyxin p CPS boiled LPS LPS+ polymyxin fl LPS boiled Unstimulated Unstimulated + polymyxin fl Media control
17.1 +6.8 11.2 + 0.9 b 23.4 + 6.1 30.6 + 6.5 12.5 + 1.4c 28.6 + 6.6 23.2 + 5.7 11.2 + 0.4 d 3.0 + 1.1
[ 3H ]-thymidine incorporation was determined and compared to thymocytes stimulated with PHA alone (3.0 + I. I cpm × 103). The results are the mean + SEM of three separate experiments. aBovine adherent monocyte were cultured as indicated for 18-20 h with 1/zg per ml, 5 pg per ml LPS or 1/zg per ml polymyxin p. IL-1 activity was detected by adding culture supernatants to C3H/HeJ mouse thymocytes stimulated with a suboptimal concentration of PHA. bp< 0.05 compared to monocytes treated with CPS. , ~ _ ,~ n¢ C~,~..p~r~rl t¢~ m c m o c v t e s t r e a t e d w i t h L P S . dp
