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Bovine alveolar macrophage neurokinin-1 and response to substance P
Veterinary Immunology and Immunopathology 112 (2006) 290–295 www.elsevier.com/locate/vetimm
Short communication
Bovine alveolar macrophage neurokinin-1 and response to substance P Donna P. Rogers a, Carol R. Wyatt a, Paul H. Walz b, James S. Drouillard c, Derek A. Mosier a,* a
Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, United States b Department of Clinical Sciences, Auburn University, AL, United States c Department of Animal Sciences, Kansas State University, Manhattan, KS, United States Received 21 December 2005; accepted 2 March 2006
Abstract In this study bovine alveolar macrophage neurokinin-1 (NK-1) and the in vitro response to substance P (SP) exposure were investigated. Bovine alveolar macrophage membrane extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using anti-NK-1 antiserum demonstrated the presence of an approximately 60 kDa band. Phagocytosis of fluorescent bioparticles by SP-exposed macrophages was 39% greater than that of non-exposed macrophages (P = 0.0089). Likewise, there was 28% greater TNF production by macrophages following SP exposure compared to non-exposed controls (P = 0.116). These results suggest that bovine alveolar macrophages respond to SP at least in part by enhancing phagocytosis and TNF production. # 2006 Elsevier B.V. All rights reserved. Keywords: Bovine; Macrophage; Substance P; Neurokinin-1
1. Introduction Substance P (SP) is a neuropeptide released from sensory C-fibers that can induce inflammatory changes such as vasodilation, increased endothelial permeability, and neutrophil adherence to endothelium and bronchial epithelial cells (Baluk et al., 1995; Barnes, 2001; McDonald et al., 1996; Payan, 1989). Nerve endings containing SP are located throughout * Corresponding author. Tel.: +1 785 532 4410. E-mail address: [email protected] (D.A. Mosier).
the body, but are most numerous in immunologically active areas such as the skin, gastrointestional tract, and respiratory tract (Pernow, 1983). In cattle SPreactive nerve endings are most numerous in the nasal, laryngeal, and tracheal mucosa, and less numerous in the lung (Nishi et al., 2000). Within the lung the SPcontaining fibers are present in pulmonary epithelium, the connective tissue beneath the epithelium and around blood vessels and glands, but are sparse in smooth muscle layers. Neurokinin-1 (NK-1), the major receptor for SP, has been identified on a variety of cell types, including
0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.03.001
D.P. Rogers et al. / Veterinary Immunology and Immunopathology 112 (2006) 290–295
endothelium, pulmonary epithelium, mast cells, neutrophils, and mononuclear cells (Chancellor-Freeland et al., 1995; Grubor et al., 2004; Ho et al., 1997). Stimulation of NK-1 on macrophages from various species enhances functions such as oxidative metabolism and the production of eicosanoid mediators, NO production, chemotaxis, and cytokine production (Chancellor-Freeland et al., 1995; Hood et al., 2000; Jeon et al., 1999; Lotz et al., 1988; McGillis et al., 1990; Murris-Espin et al., 1995). The presence of NK1 and specific effects of SP on bovine alveolar macrophages have not been reported. In this study we identify the presence of NK-1 on bovine alveolar macrophages, and investigate two important effects of SP stimulation on these cells.
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Macrophage viability was approximately 90% as assessed by trypan blue exclusion (Sigma–Aldrich, St. Louis, MO). Washed macrophages from seven heifers were suspended in Dulbecco’s Minimal Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) containing 2% FBS for phagocytosis and TNF assays. Lavage samples from three heifers were processed for SDSPAGE and immunoblotting. Macrophage membranes were extracted using a lysis buffer containing 1% Triton-X-114. Following incubation at 37 8C, the membrane phase was removed, treated with protease inhibitor and incubated overnight with 100% ethanol at 20 8C to further precipitate the protein (Hsu-Fong et al., 2003). 2.3. Antibodies
Alveolar macrophages were obtained from 10 healthy yearling cross-bred heifers. Heifers were purchased for a feeding trial at the Kansas State University Beef Teaching and Research Center. Heifers were housed at the Beef Teaching and Research Center, fed a corn, corn silage, prairie hay mix to meet National Research Council requirements, and had unlimited access to water. The experimental protocol was approved by the Kansas State University Institutional Animal Care and Use Committee.
A synthetic SP receptor NK-1 peptide was injected into guinea pigs for production of antiserum for use in immunoblotting experiments (Chemicon, International, Temecula, CA). The anti-bovine TNF was derived from a commercially produced recombinant TNF protein (ATG Laboratories, Eden Prairie, MN). The recombinant protein (10 ng/mL) was confirmed to have biological activity of TNF by measuring the cytolytic effect of the recombinant protein on murine WEHI-13VAR-164 cells in the presence of actinomycin D. The recombinant TNF protein was conjugated to keyhole limpet hemocyanin and was then injected into goats for antibody production. Polyclonal antibody was harvested and affinity purified against the recombinant TNF protein.
2.2. Alveolar macrophage preparation
2.4. SDS-PAGE and immunoblotting
Alveolar macrophages were obtained by bronchoalveolar lavage. A polyethylene tube (6.4 mm diameter with a 7.9 mm diameter rubber tip) was passed intranasally and lodged in a small diameter airway. One hundred-twenty milliliters of phosphatebuffered saline (PBS) were introduced into the lung, and retrieved by aspiration back through the tube. Lavage samples were centrifuged and cell pellets were washed three times with Hanks’ balanced salt solution without Ca2+ and Mg2+ (HBSS; Invitrogen, Carlsbad, CA). Bronchoalveolar lavages contained, on average, 80% macrophages, as determined by flow cytometric forward and side scatter.
Macrophage membrane suspensions were diluted with Laemmli sample buffer (1:2; Bio-Rad, San Francisco, CA) and 40 mL were loaded onto a 12% SDS-PAGE gel (Bio-Rad, San Francisco, CA). A molecular weight marker (Amersham BioSciences, Piscataway, NJ) was included. Proteins transferred to nitrocellulose membrane were blocked overnight with PBS-Tween 20 (PBST) containing 2% gelatin, reacted against a 1:200 dilution of primary antibody anti-NK-1 (guinea pig) and a 1:400 dilution of secondary antibody consisting of horseradish peroxidase-labeled goat anti-guinea pig (KPL, Gaithersburg, MD).
2. Materials and methods 2.1. Animals
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D.P. Rogers et al. / Veterinary Immunology and Immunopathology 112 (2006) 290–295
2.5. Phagocytosis assay Phagocytosis was evaluated using a commercial assay kit (Vybrant Phagocytosis Assay Kit, Molecular Probes, Eugene, OR). Alveolar macrophages (3– 5 105 well 1) in 100 mL DMEM were placed in 96well cell culture plates and incubated overnight in 5% CO2 at 37 8C. To each well, either 50 mL of DMEM (negative control), 10 9 M N-formyl-Met-Leu-Phe (fMLP; Sigma–Aldrich, St. Louis, MO) (positive control), or 10 9 M SP (Sigma–Aldrich, St. Louis, MO) were added followed by a 1 h incubation as before. Supernatants were then removed and stored at 20 8C for use in TNF assays. One hundred microliters of HBSS solution containing fluorescent Escherichia coli bioparticles were added to the macrophage pellet in each well and incubated for 2 h as before. Supernatants containing remaining bioparticles were aspirated from the wells, and 100 mL of trypan blue were added to each well to quench extracellular fluorescence, and incubated for 1 min at room temperature. Trypan blue was removed by aspiration, and the wells were measured for fluorescence on a fluorescent plate reader at 480 nm excitation, 520 nm emission (Fluoroskan Ascent FL, LabSystems, Franklin, MA). Fluorescence data for experimental groups were compared by Student’s ttests. 2.6. TNF assay PBS-saturated nitrocellulose was placed in a dot blot apparatus (Bio-Dot, Biorad, San Francisco, CA), and dried by vacuum aspiration. One hundred microliters of culture supernatants obtained previously from macrophages treated with either DMEM, fMLP, or SP were placed in individual wells of the apparatus and incubated at room temperature for 40 min. One hundred microliters of PBST containing 1% FBS were added and incubation continued for 20 more minutes. Following incubation, wells were washed twice with 200 mL PBST, and 100 mL of primary antibody consisting of a 1:10 dilution of goat polyclonal antibody against bovine TNF were added and incubated for 30 min. Wells were washed three times with 200 mL PBST, and 100 mL of a 1:50 dilution of secondary antibody consisting of horseradish peroxidase-labeled rabbit anti-goat antibody
(EY Laboratories, San Mateo, CA), were added and incubated as before. Following three washes with 200 mL PBST, the nitrocellulose was reacted with a 4chloro-1-naphthol: hydrogen peroxidase: methanol color reagent. Dot density was determined by comparison with a gray scale standard ranging from 255 units (white, no reactivity) to 0 units (black, maximum reactivity) (Adobe Photoshop 5.0 LE, Adobe Systems Inc, San Jose, CA). Density was used as an estimate of the relative amount of TNF in each sample. The TNF values for experimental groups were compared by Student’s t-tests.
3. Results and discussion Alveolar macrophages play a prominent role in the pathogenesis of pneumonia through activities such as phagocytosis and the production of proinflammatory substances such as reactive oxygen metabolites and cytokines (Sibille and Reynolds, 1990). The purpose of this study was to determine if SP could influence these activities in bovine alveolar macrophages by first demonstrating that the high affinity SP receptor NK-1 is present on bovine alveolar macrophages, and secondly if exposure to SP influences phagocytosis or TNF production, presumably by stimulation of NK-1. Similar to macrophages from other species, NK-1 was identified in membrane extracts of bovine alveolar macrophages by anti-NK-1 antibody (Fig. 1) (Chancellor-Freeland et al., 1995; Grubor et al., 2004; Ho et al., 1997). When SP-exposed and non-exposed bovine alveolar macrophages were incubated with fluorescent bioparticles, there was a 39% greater mean fluorescence for SP-exposed macrophages compared to the mean of media-only controls (P = 0.0089) (Table 1). Alveolar macrophages exposed to fMLP had a 58% increase in mean fluorescence compared to the media-only controls (P = 0.0127). There was only an 8% difference in mean fluorescence between fMLP and SP media controls (P = 0.5439), and a 6% difference in mean fluorescence between fMLP and SP-exposed macrophages (P = 0.5659). Cytokine production within the lungs is a particularly important contributor to inflammation in pneumonia, including pneumonia in cattle caused by infection with M. haemolytica (bovine pneumonic pasteurellosis) (van der Sluijs et al., 2004; Yoo et al.,
D.P. Rogers et al. / Veterinary Immunology and Immunopathology 112 (2006) 290–295
Fig. 1. SDS-PAGE and immunoblotting of membrane extracts of bovine alveolar macrophages. A single approximately 60 kDa band was detected by guinea pig anti-NK-1 primary antibody.
1995). Exposure of bovine alveolar macrophages to M. haemolytica in vitro caused increased synthesis of IL-1, TNF, and IL-8 mRNA (Morsey et al., 1999). Similarly, production of these cytokines by alveolar macrophages and other pulmonary cells was increased
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in vivo following experimental challenge of calves with M. haemolytica (Yoo et al., 1995; Malazdrewich et al., 2001). In the current study SP exposure was also associated with increased TNF production by bovine alveolar macrophages. There was a 28% increase in mean TNF production by SP-exposed samples compared to non-exposed control samples (P = 0.116) (Table 1). Similarly, there was a 32% increase in mean TNF production for fMLP-exposed samples compared to non-exposed control samples (P = 0.158). There was only a 5% difference in mean TNF values between the fMLP and SP controls (P = 0.755), and a 1% difference in mean TNF values between the fMLP- and SPexposed samples (P = 0.885). These results are similar to the overall stimulatory effect of SP on macrophages from other species (Hood et al., 2000; Jeon et al., 1999; Lotz et al., 1988; McGillis et al., 1990; Murris-Espin et al., 1995). The demonstration of stimulatory effects of SP on bovine alveolar macrophages indicates the potential for SP to act synergistically with M. haemolytica lipopolysaccharide (LPS) and leukotoxin (LKT), the major virulence factors involved in the pathogenesis of pneumonic pasteurellosis (Confer et al., 1990). Following co-stimulation with LPS, low concentrations of SP had potent proinflammatory effects on human leukocytes and neuroglial cells (Luber-Narod et al., 1994; Perianin et al., 1989). Lipopolysaccharide priming enhanced SP-induced neutrophil adherence to pulmonary epithelium, and the combination of SP and LPS significantly increased IL-1 and TNF release by human neutrophils and pulmonary epithelium (Kuo et al., 2000). These effects appeared to be due to an enhancement of the NK-1 receptor by LPS either through upregulating NK-1 expression, increasing affinity for NK-1, or through a synergism between the
Table 1 Phagocytosis of fluorescent bioparticlesa and TNF productionb by N-formyl-Met-Leu-Phe and substance P-exposed and non-exposed bovine alveolar macrophages fMLPd control non-exposed a
Phagocytosis TNF productionb a
c
1.2 1.0 32.1 9.9 c
fMLP exposed
SPd control non-exposed
SP exposed
1.9 1.4 42.2 13.2
1.3 1.2 33.6 11.0
1.8 1.4 42.8 5.2
Fluorescence of bovine alveolar macrophages following phagocytosis of fluorescent bioparticles (480 nm excitation, 520 nm emission). Dot-blot density of bovine alveolar macrophage supernatants reacted with anti-TNF antibody quantitated on a gray scale spectrum with a range of 0 (white, maximum reactivity) to 255 (black, minimum reactivity). Reported values are percent reactivity [100-scale value (100/ 255)]. c Mean S.D., n = 7. d fMLP = N-formyl-Met-Leu-Phe, SP = Substance P. b
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independent stimulation of NK-1 by SP and LPS. Similarly, SP priming of mouse peritoneal macrophages prior to exposure to LPS resulted in significant enhancement of IL-1, IL-6 and TNF secretion compared to LPS alone (Berman et al., 1996). M. haemolytica LKT mediates its cytotoxicity for bovine leukocytes by binding to CD11a/CD18 (Ambagala et al., 1999; Jeyaseelan et al., 2000; Li et al., 1999). Increased expression of neutrophil CD11a/CD18 has been associated with increased LKT binding and increased neutrophil cytotoxicity (Leite et al., 2000; Leite et al., 2002). Upregulation of neutrophil CD11b/CD18 expression by neutrophils exposed to a combination of LPS and SP was reported as one mechanism of enhanced neutrophil adhesion to pulmonary epithelium (Kuo et al., 2000). Synergism between SP and LPS may be another mechanism that could enhance the cytotoxic effect of LKT by increasing the number of CD11a/CD18 receptors on bovine leukocytes. Acknowledgements The authors thank Jennifer Waite and Susie Larson for assistance with manuscript preparation. This work was supported in part by funds from the Kansas Agricultural Experiment Station. Published as contribution #05-67-J of the KAES. References Ambagala, T.C., Ambagala, A.P., Srikumaran, S., 1999. The leukotoxin of Pasteurella haemolytica binds to beta(2) integrins on bovine leukocytes. FEMS Microbiol. Lett. 179, 161–167. Baluk, P., Bertrand, C., Geppetti, P., et al., 1995. NK 1 receptors mediate leukocyte adhesion in neurogenic inflammation in rat trachea. Am. J. Physiol. 268, L263–L269. Barnes, P.J., 2001. Neurogenic inflammation in the airways. Respir. Physiol. 125, 145–154. Berman, A.S., Chancellor-Freeland, C.C., Zhu, G., et al., 1996. Substance P primes murine peritoneal macrophages for an augmented proinflammatory cytokine response to lipopolysaccharide. Neuroimmunomodulation 3, 141–149. Chancellor-Freeland, C., Zhu, G.F., Kage, R., et al., 1995. Substance P and stress-induced changes in macrophages. Ann. N.Y. Acad. Sci. 771, 472–484. Confer, A.W., Panciera, R.J., Clinkenbeard, K.D., et al., 1990. Molecular aspects of virulence of Pasteurella haemolytica. Can. J. Vet. Res. 54, S48–S52.
Grubor, B., Ramirez-Romero, R., Gallup, J.M., et al., 2004. Distribution of substance P receptor (Neurokinin-1 receptor) in normal ovine lung and during the progression of bronchopneumonia in sheep. J. Histochem. Cytochem. 52, 123–130. Ho, W.Z., Lai, J.P., Zhu, X.H., et al., 1997. Human monocytes and macrophages express substance P and neurokinin-1 receptor. J. Immunol. 159, 5654–5660. Hood, V.C., Cruwys, S.C., Urban, L., et al., 2000. Differential role of neurokinin receptors in human lymphocyte and monocyte chemotaxis. Regul. Peptides 96, 17–21. Hsu-Fong, W., John, B.S., Thomas, E.B., Douglas, J., Carol, R.W., 2003. Detection of antibodies to a recombinant Cryptosporidium parvum P23 in serum and feces from neonatal calves. J. Parasitol. 89 (5), 918–923. Jeon, H.K., Jung, N.P., Choi, I.H., et al., 1999. Substance P augments nitric oxide production and gene expression in murine macrophages. Immunopharmacology 41, 219–226. Jeyaseelan, S., Hsuan, S.L., Kannan, M.S., et al., 2000. Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes. Infect. Immun. 68, 72–79. Kuo, H.P., Lin, H.C., Hwang, K.H., et al., 2000. Lipopolysaccharide enhances substance P-mediated neutrophil adherence to epithelial cells and cytokine release. Am. J. Respir. Crit. Care Med. 162, 1891–1897. Leite, F., Brown, J.F., Sylte, M.J., et al., 2000. Recombinant bovine interleukin-1b amplifies the effects of partially purified Pasteurella haemolytica leukotoxin on bovine neutrophils in a b2integrin-dependent manner. Infect. Immun. 68, 5581–5586. Leite, F., O’Brien, S., Sylte, M.J., et al., 2002. Inflammatory cytokines enhance the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood neutrophils in vitro. Infect. Immun. 70, 4336–4343. Li, J., Clinkenbeard, K.D., Ritchey, J.W., 1999. Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin. Vet. Microbiol. 67, 91–97. Lotz, M., Vaughan, J.H., Crason, D.A., 1988. Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science 241, 1218–1220. Luber-Narod, J., Kage, R., Leeman, S.E., 1994. Substance P enhances the secretion of tumor necrosis factor-alpha from neuroglial cells stimulated with lipopolysaccharide. J. Immunol. 152, 819–824. Malazdrewich, C., Ames, T.R., Abrahamsen, M.S., et al., 2001. Pulmonary expression of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-8 in the acute phase of bovine pneumonic pasteurellosis. Vet. Pathol. 38, 297–310. McDonald, D.M., Bowden, J.J., Baluk, P., et al., 1996. Neurogenic inflammation. A model for studying efferent actions of sensory nerves. Adv. Exp. Med. Biol. 410, 453–462. McGillis, J.P., Mitsuhashi, M., Payan, D.G., 1990. Immunomoduation by tachykinin neuropeptides. Ann. N.Y. Acad. Sci. 594, 85– 94. Morsey, M.A., Van-Kessel, A.G., Mori, Y., et al., 1999. Cytokine profiles following interaction between bovine alveolar macrophages and Pasteurella haemolytica. Microb. Pathog. 26, 325– 331.
D.P. Rogers et al. / Veterinary Immunology and Immunopathology 112 (2006) 290–295 Murris-Espin, M., Pinelli, E., Pipy, B., et al., 1995. Substance P and alveolar macrophages: effects on oxidative metabolism and eicosanoid production. Allergy 50, 334–339. Nishi, Y., Kitamura, N., Otani, M., et al., 2000. Distribution of capsaicin-sensitive substance P- and calcitonin gene-related peptide-immunoreactive nerves in bovine respiratory tract. Anat. Anz. 182, 319–326. Payan, D.G., 1989. Neuropeptides and inflammation: the role of substance P. Annu. Rev. Med. 40, 341–352. Perianin, A., Snyderman, R., Malfroy, B., 1989. Substance P primes human neutrophil activation: a mechanism for neurological regulation of inflammation. Biochem. Biophys. Res. Commun. 161, 520–524.
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Pernow, B., 1983. Substance P. Pharmacol. Rev. 35, 85–141. Sibille, Y., Reynolds, H.Y., 1990. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am. Rev. Respir. Dis. 141, 471–501. van der Sluijs, K., van Elden, L.F.R., Nijhuis, M., et al., 2004. IL-10 is an important mediator of the enhanced susceptibility to pneumococcal pneumonia after influenza infection. J. Immunol. 172, 7603–7609. Yoo, H.S., Maheswaran, S.K., Srinand, S., et al., 1995. Increased tumor necrosis factor-alpha and interleukin-1 beta expression in the lungs of calves with experimental pneumonic pasteurellosis. Vet. Immunol. Immunopathol. 49, 15–28.
Short communication
Bovine alveolar macrophage neurokinin-1 and response to substance P Donna P. Rogers a, Carol R. Wyatt a, Paul H. Walz b, James S. Drouillard c, Derek A. Mosier a,* a
Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, United States b Department of Clinical Sciences, Auburn University, AL, United States c Department of Animal Sciences, Kansas State University, Manhattan, KS, United States Received 21 December 2005; accepted 2 March 2006
Abstract In this study bovine alveolar macrophage neurokinin-1 (NK-1) and the in vitro response to substance P (SP) exposure were investigated. Bovine alveolar macrophage membrane extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using anti-NK-1 antiserum demonstrated the presence of an approximately 60 kDa band. Phagocytosis of fluorescent bioparticles by SP-exposed macrophages was 39% greater than that of non-exposed macrophages (P = 0.0089). Likewise, there was 28% greater TNF production by macrophages following SP exposure compared to non-exposed controls (P = 0.116). These results suggest that bovine alveolar macrophages respond to SP at least in part by enhancing phagocytosis and TNF production. # 2006 Elsevier B.V. All rights reserved. Keywords: Bovine; Macrophage; Substance P; Neurokinin-1
1. Introduction Substance P (SP) is a neuropeptide released from sensory C-fibers that can induce inflammatory changes such as vasodilation, increased endothelial permeability, and neutrophil adherence to endothelium and bronchial epithelial cells (Baluk et al., 1995; Barnes, 2001; McDonald et al., 1996; Payan, 1989). Nerve endings containing SP are located throughout * Corresponding author. Tel.: +1 785 532 4410. E-mail address: [email protected] (D.A. Mosier).
the body, but are most numerous in immunologically active areas such as the skin, gastrointestional tract, and respiratory tract (Pernow, 1983). In cattle SPreactive nerve endings are most numerous in the nasal, laryngeal, and tracheal mucosa, and less numerous in the lung (Nishi et al., 2000). Within the lung the SPcontaining fibers are present in pulmonary epithelium, the connective tissue beneath the epithelium and around blood vessels and glands, but are sparse in smooth muscle layers. Neurokinin-1 (NK-1), the major receptor for SP, has been identified on a variety of cell types, including
0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.03.001
D.P. Rogers et al. / Veterinary Immunology and Immunopathology 112 (2006) 290–295
endothelium, pulmonary epithelium, mast cells, neutrophils, and mononuclear cells (Chancellor-Freeland et al., 1995; Grubor et al., 2004; Ho et al., 1997). Stimulation of NK-1 on macrophages from various species enhances functions such as oxidative metabolism and the production of eicosanoid mediators, NO production, chemotaxis, and cytokine production (Chancellor-Freeland et al., 1995; Hood et al., 2000; Jeon et al., 1999; Lotz et al., 1988; McGillis et al., 1990; Murris-Espin et al., 1995). The presence of NK1 and specific effects of SP on bovine alveolar macrophages have not been reported. In this study we identify the presence of NK-1 on bovine alveolar macrophages, and investigate two important effects of SP stimulation on these cells.
291
Macrophage viability was approximately 90% as assessed by trypan blue exclusion (Sigma–Aldrich, St. Louis, MO). Washed macrophages from seven heifers were suspended in Dulbecco’s Minimal Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) containing 2% FBS for phagocytosis and TNF assays. Lavage samples from three heifers were processed for SDSPAGE and immunoblotting. Macrophage membranes were extracted using a lysis buffer containing 1% Triton-X-114. Following incubation at 37 8C, the membrane phase was removed, treated with protease inhibitor and incubated overnight with 100% ethanol at 20 8C to further precipitate the protein (Hsu-Fong et al., 2003). 2.3. Antibodies
Alveolar macrophages were obtained from 10 healthy yearling cross-bred heifers. Heifers were purchased for a feeding trial at the Kansas State University Beef Teaching and Research Center. Heifers were housed at the Beef Teaching and Research Center, fed a corn, corn silage, prairie hay mix to meet National Research Council requirements, and had unlimited access to water. The experimental protocol was approved by the Kansas State University Institutional Animal Care and Use Committee.
A synthetic SP receptor NK-1 peptide was injected into guinea pigs for production of antiserum for use in immunoblotting experiments (Chemicon, International, Temecula, CA). The anti-bovine TNF was derived from a commercially produced recombinant TNF protein (ATG Laboratories, Eden Prairie, MN). The recombinant protein (10 ng/mL) was confirmed to have biological activity of TNF by measuring the cytolytic effect of the recombinant protein on murine WEHI-13VAR-164 cells in the presence of actinomycin D. The recombinant TNF protein was conjugated to keyhole limpet hemocyanin and was then injected into goats for antibody production. Polyclonal antibody was harvested and affinity purified against the recombinant TNF protein.
2.2. Alveolar macrophage preparation
2.4. SDS-PAGE and immunoblotting
Alveolar macrophages were obtained by bronchoalveolar lavage. A polyethylene tube (6.4 mm diameter with a 7.9 mm diameter rubber tip) was passed intranasally and lodged in a small diameter airway. One hundred-twenty milliliters of phosphatebuffered saline (PBS) were introduced into the lung, and retrieved by aspiration back through the tube. Lavage samples were centrifuged and cell pellets were washed three times with Hanks’ balanced salt solution without Ca2+ and Mg2+ (HBSS; Invitrogen, Carlsbad, CA). Bronchoalveolar lavages contained, on average, 80% macrophages, as determined by flow cytometric forward and side scatter.
Macrophage membrane suspensions were diluted with Laemmli sample buffer (1:2; Bio-Rad, San Francisco, CA) and 40 mL were loaded onto a 12% SDS-PAGE gel (Bio-Rad, San Francisco, CA). A molecular weight marker (Amersham BioSciences, Piscataway, NJ) was included. Proteins transferred to nitrocellulose membrane were blocked overnight with PBS-Tween 20 (PBST) containing 2% gelatin, reacted against a 1:200 dilution of primary antibody anti-NK-1 (guinea pig) and a 1:400 dilution of secondary antibody consisting of horseradish peroxidase-labeled goat anti-guinea pig (KPL, Gaithersburg, MD).
2. Materials and methods 2.1. Animals
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D.P. Rogers et al. / Veterinary Immunology and Immunopathology 112 (2006) 290–295
2.5. Phagocytosis assay Phagocytosis was evaluated using a commercial assay kit (Vybrant Phagocytosis Assay Kit, Molecular Probes, Eugene, OR). Alveolar macrophages (3– 5 105 well 1) in 100 mL DMEM were placed in 96well cell culture plates and incubated overnight in 5% CO2 at 37 8C. To each well, either 50 mL of DMEM (negative control), 10 9 M N-formyl-Met-Leu-Phe (fMLP; Sigma–Aldrich, St. Louis, MO) (positive control), or 10 9 M SP (Sigma–Aldrich, St. Louis, MO) were added followed by a 1 h incubation as before. Supernatants were then removed and stored at 20 8C for use in TNF assays. One hundred microliters of HBSS solution containing fluorescent Escherichia coli bioparticles were added to the macrophage pellet in each well and incubated for 2 h as before. Supernatants containing remaining bioparticles were aspirated from the wells, and 100 mL of trypan blue were added to each well to quench extracellular fluorescence, and incubated for 1 min at room temperature. Trypan blue was removed by aspiration, and the wells were measured for fluorescence on a fluorescent plate reader at 480 nm excitation, 520 nm emission (Fluoroskan Ascent FL, LabSystems, Franklin, MA). Fluorescence data for experimental groups were compared by Student’s ttests. 2.6. TNF assay PBS-saturated nitrocellulose was placed in a dot blot apparatus (Bio-Dot, Biorad, San Francisco, CA), and dried by vacuum aspiration. One hundred microliters of culture supernatants obtained previously from macrophages treated with either DMEM, fMLP, or SP were placed in individual wells of the apparatus and incubated at room temperature for 40 min. One hundred microliters of PBST containing 1% FBS were added and incubation continued for 20 more minutes. Following incubation, wells were washed twice with 200 mL PBST, and 100 mL of primary antibody consisting of a 1:10 dilution of goat polyclonal antibody against bovine TNF were added and incubated for 30 min. Wells were washed three times with 200 mL PBST, and 100 mL of a 1:50 dilution of secondary antibody consisting of horseradish peroxidase-labeled rabbit anti-goat antibody
(EY Laboratories, San Mateo, CA), were added and incubated as before. Following three washes with 200 mL PBST, the nitrocellulose was reacted with a 4chloro-1-naphthol: hydrogen peroxidase: methanol color reagent. Dot density was determined by comparison with a gray scale standard ranging from 255 units (white, no reactivity) to 0 units (black, maximum reactivity) (Adobe Photoshop 5.0 LE, Adobe Systems Inc, San Jose, CA). Density was used as an estimate of the relative amount of TNF in each sample. The TNF values for experimental groups were compared by Student’s t-tests.
3. Results and discussion Alveolar macrophages play a prominent role in the pathogenesis of pneumonia through activities such as phagocytosis and the production of proinflammatory substances such as reactive oxygen metabolites and cytokines (Sibille and Reynolds, 1990). The purpose of this study was to determine if SP could influence these activities in bovine alveolar macrophages by first demonstrating that the high affinity SP receptor NK-1 is present on bovine alveolar macrophages, and secondly if exposure to SP influences phagocytosis or TNF production, presumably by stimulation of NK-1. Similar to macrophages from other species, NK-1 was identified in membrane extracts of bovine alveolar macrophages by anti-NK-1 antibody (Fig. 1) (Chancellor-Freeland et al., 1995; Grubor et al., 2004; Ho et al., 1997). When SP-exposed and non-exposed bovine alveolar macrophages were incubated with fluorescent bioparticles, there was a 39% greater mean fluorescence for SP-exposed macrophages compared to the mean of media-only controls (P = 0.0089) (Table 1). Alveolar macrophages exposed to fMLP had a 58% increase in mean fluorescence compared to the media-only controls (P = 0.0127). There was only an 8% difference in mean fluorescence between fMLP and SP media controls (P = 0.5439), and a 6% difference in mean fluorescence between fMLP and SP-exposed macrophages (P = 0.5659). Cytokine production within the lungs is a particularly important contributor to inflammation in pneumonia, including pneumonia in cattle caused by infection with M. haemolytica (bovine pneumonic pasteurellosis) (van der Sluijs et al., 2004; Yoo et al.,
D.P. Rogers et al. / Veterinary Immunology and Immunopathology 112 (2006) 290–295
Fig. 1. SDS-PAGE and immunoblotting of membrane extracts of bovine alveolar macrophages. A single approximately 60 kDa band was detected by guinea pig anti-NK-1 primary antibody.
1995). Exposure of bovine alveolar macrophages to M. haemolytica in vitro caused increased synthesis of IL-1, TNF, and IL-8 mRNA (Morsey et al., 1999). Similarly, production of these cytokines by alveolar macrophages and other pulmonary cells was increased
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in vivo following experimental challenge of calves with M. haemolytica (Yoo et al., 1995; Malazdrewich et al., 2001). In the current study SP exposure was also associated with increased TNF production by bovine alveolar macrophages. There was a 28% increase in mean TNF production by SP-exposed samples compared to non-exposed control samples (P = 0.116) (Table 1). Similarly, there was a 32% increase in mean TNF production for fMLP-exposed samples compared to non-exposed control samples (P = 0.158). There was only a 5% difference in mean TNF values between the fMLP and SP controls (P = 0.755), and a 1% difference in mean TNF values between the fMLP- and SPexposed samples (P = 0.885). These results are similar to the overall stimulatory effect of SP on macrophages from other species (Hood et al., 2000; Jeon et al., 1999; Lotz et al., 1988; McGillis et al., 1990; Murris-Espin et al., 1995). The demonstration of stimulatory effects of SP on bovine alveolar macrophages indicates the potential for SP to act synergistically with M. haemolytica lipopolysaccharide (LPS) and leukotoxin (LKT), the major virulence factors involved in the pathogenesis of pneumonic pasteurellosis (Confer et al., 1990). Following co-stimulation with LPS, low concentrations of SP had potent proinflammatory effects on human leukocytes and neuroglial cells (Luber-Narod et al., 1994; Perianin et al., 1989). Lipopolysaccharide priming enhanced SP-induced neutrophil adherence to pulmonary epithelium, and the combination of SP and LPS significantly increased IL-1 and TNF release by human neutrophils and pulmonary epithelium (Kuo et al., 2000). These effects appeared to be due to an enhancement of the NK-1 receptor by LPS either through upregulating NK-1 expression, increasing affinity for NK-1, or through a synergism between the
Table 1 Phagocytosis of fluorescent bioparticlesa and TNF productionb by N-formyl-Met-Leu-Phe and substance P-exposed and non-exposed bovine alveolar macrophages fMLPd control non-exposed a
Phagocytosis TNF productionb a
c
1.2 1.0 32.1 9.9 c
fMLP exposed
SPd control non-exposed
SP exposed
1.9 1.4 42.2 13.2
1.3 1.2 33.6 11.0
1.8 1.4 42.8 5.2
Fluorescence of bovine alveolar macrophages following phagocytosis of fluorescent bioparticles (480 nm excitation, 520 nm emission). Dot-blot density of bovine alveolar macrophage supernatants reacted with anti-TNF antibody quantitated on a gray scale spectrum with a range of 0 (white, maximum reactivity) to 255 (black, minimum reactivity). Reported values are percent reactivity [100-scale value (100/ 255)]. c Mean S.D., n = 7. d fMLP = N-formyl-Met-Leu-Phe, SP = Substance P. b
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independent stimulation of NK-1 by SP and LPS. Similarly, SP priming of mouse peritoneal macrophages prior to exposure to LPS resulted in significant enhancement of IL-1, IL-6 and TNF secretion compared to LPS alone (Berman et al., 1996). M. haemolytica LKT mediates its cytotoxicity for bovine leukocytes by binding to CD11a/CD18 (Ambagala et al., 1999; Jeyaseelan et al., 2000; Li et al., 1999). Increased expression of neutrophil CD11a/CD18 has been associated with increased LKT binding and increased neutrophil cytotoxicity (Leite et al., 2000; Leite et al., 2002). Upregulation of neutrophil CD11b/CD18 expression by neutrophils exposed to a combination of LPS and SP was reported as one mechanism of enhanced neutrophil adhesion to pulmonary epithelium (Kuo et al., 2000). Synergism between SP and LPS may be another mechanism that could enhance the cytotoxic effect of LKT by increasing the number of CD11a/CD18 receptors on bovine leukocytes. Acknowledgements The authors thank Jennifer Waite and Susie Larson for assistance with manuscript preparation. This work was supported in part by funds from the Kansas Agricultural Experiment Station. Published as contribution #05-67-J of the KAES. References Ambagala, T.C., Ambagala, A.P., Srikumaran, S., 1999. The leukotoxin of Pasteurella haemolytica binds to beta(2) integrins on bovine leukocytes. FEMS Microbiol. Lett. 179, 161–167. Baluk, P., Bertrand, C., Geppetti, P., et al., 1995. NK 1 receptors mediate leukocyte adhesion in neurogenic inflammation in rat trachea. Am. J. Physiol. 268, L263–L269. Barnes, P.J., 2001. Neurogenic inflammation in the airways. Respir. Physiol. 125, 145–154. Berman, A.S., Chancellor-Freeland, C.C., Zhu, G., et al., 1996. Substance P primes murine peritoneal macrophages for an augmented proinflammatory cytokine response to lipopolysaccharide. Neuroimmunomodulation 3, 141–149. Chancellor-Freeland, C., Zhu, G.F., Kage, R., et al., 1995. Substance P and stress-induced changes in macrophages. Ann. N.Y. Acad. Sci. 771, 472–484. Confer, A.W., Panciera, R.J., Clinkenbeard, K.D., et al., 1990. Molecular aspects of virulence of Pasteurella haemolytica. Can. J. Vet. Res. 54, S48–S52.
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