INFEcriON AND IMMUNrrY, Mar. 1975, p. 488-492 Copyright 0 1975 American Society for Microbiology
Vol. 11. No. 3 Printed in U.S.A.
Quantification of Mouse Macrophage Chemotaxis in Vitro: Role of C5 for the Production of Chemotactic Activity RALPH SNYDERMAN,* MARILYN C. PIKE, DEAN McCARLEY, AND LAWRENCE LANG Division of Rheumatic and Genetic Disease, Departments of Medicine and Immunology, Duke University Medical Center, Durham, North Carolina 27710 Received for publication 27 November 1974
Delineation of the mechanisms of macrophage accumulation at local tissue sites will further our understanding of immunologically mediated host resistance to infectious and neoplastic diseases. Since mice are frequently used for the study of immune function, we developed a method for the quantification of mouse macrophage chemotaxis in vitro. By this method it was found that the fifth component of complement is necessary for the production of chemotactic activity in mouse serum by inflammatory agents such as endotoxin or aggregated gamma globulin. The majority of macrophage chemotactic activity produced by these agents in mouse serum can be attributed to a low-molecular-weight (ca. 15,000) chemotactic factor. The data suggest that this factor is the biologically active cleavage product of the fifth component of complement, C5a.
Accumulation of macrophages at local tissue sites is an important event in wound healing and in immunologically mediated host defense. Methods for the quantification of macrophage migration can therefore provide valuable tools for the study of factors involved in host resistance to infectious and neoplastic diseases. The availability of quantitative methods for the study of leukocyte chemotaxis in vitro (2, 10, 14, 15, 18) has resulted in the characterization of several chemotactic factors as well as the delineation of abnormalities of leukotaxis in certain human disease states (9, 18, 19, 22). Although macrophage chemotaxis assays have been developed for a number of animal species (1, 10, 14, 20), no method has been described to measure mouse macrophage migration in vitro. Since many studies of immunological parameters of host resistance are performed in mice, we developed a quantitative, reproducible assay for mouse macrophage chemotaxis and characterized factors in mouse serum which are chemotactic for macrophages in vitro.
Macrophages for the chemotaxis assay.
C3H/HeJ mice were injected intraperitoneally with
MATERIALS AND METHODS Mice. Male mice of the lines C3H/HeJ and CBA/J (C5 normal) and AKR/J and A/J (C5 deficient) (3, 4, 8) were purchased from Jackson Laboratories, Bar Harbor, Me. Mouse Serum. Blood from normal and C5-deficient mice was obtained from the retro-orbital plexus or by cardiac puncture and allowed to clot at room temperature for 30 min. The serum was removed after centrifugation and stored at -70 C until use. 488
2.0 ml of 10% proteose peptone (Difco Laboratories, Detroit, Mich.) in water 3 or 4 days before sacrifice. Mice were sacrificed by CO, asphyxiation and the peritoneal cavities were exposed by abdominal incision. The cavities were then washed with approximately 7.5 ml of Gey's balanced salt solution, pH 7.0 (Flow Laboratories, Rockville, Md.), containing 2% bovine serum albumin, 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (Calbiochem, La Jolla, Calif.) (Gey's medium), and. 10 U of heparin/ml. The peritoneal exudates from several mice were pooled and centrifuged (300 x g) at 4 C for 10 min. Approximately 8 x 10" leukocytes were obtained per mouse, and of these about 80% were macrophages and 20% were lymphocytes. The cells were washed one time in Gey's medium, and resuspended to contain 1.5 x 106 viable macrophages/ml for use in the chemotaxis assay. Viability was determined by trypan blue exclusion and generally at least 97% of the cells were viable when collected by these methods. Macrophages chemotaxis assay. Various materials were tested for chemotactic activity in vitro using a modification of methods described previously (14). In brief, the chemotactic stimulant under study was placed in the lower compartment of a modified Boyden chamber (Fig. 1) and separated by a 5-tsm polycarbonate (Nuclepore) filter (Wallabs, San Rafael, Calif.) from the upper compartment containing the cell suspension. The upper compartment of the chemotaxis chamber (Duke University Surgical Instrument Shop, Durham, N. C.) holds 0.4 ml of the cell suspension, while the lower compartment holds 0.85 ml of the chemotactic or control stimulant. All assays were performed in triplicate and the chambers
VOL. 11, 1975
MOUSE MACROPHAGE CHEMOTAXIS IN VITRO
Upper Chomber
1F~~~~~~~~~ Ite Lower
Chamber
FiG. 1. Chemotaxis chamber. A standardized cell suspension (0.4 ml) is placed in the upper compartment of the chamber and is separated from the chemotactic stimulant (0.85 ml) or medium alone in the lower compartment by a 5-,gm pore size polycarbonate or nitrocellulose filter. containing macrophages and stimulants were incubated for 4 h in humidified air at 37 C. After incubation, the chambers were emptied and the filters were removed; then fixed in ethanol for 15 s, stained for 6 min in Mayer's hematoxylin, 1 min in acid alcohol, and 1 min in bluing solution (11). Chemotaxis was quantified by counting and averaging the macrophages in 20 oil immersion fields, (x 1,540) that migrated completely through the filter. PMN chemotaxis assay. Mouse polymorphonuclear leukocytes (PMN) were isolated as described previously (13). Briefly, C3H/HeJ mice were bled by cardiac puncture into syringes containing approximately 20 U of heparin per ml of blood. The blood was mixed with an equal volume of 3% dextran (Dextran T 250, Pharmacia Fine Chemicals, Uppsala, Sweden) in normal saline. The erythrocytes were allowed to sediment for 30 min at room temperature and the leukocyte-rich plasma was withdrawn. The plasma was then centrifuged at 300 x g for 10 min and the contaminating erythrocytes were removed by rapid (20 s) hypotonic (0.2% NaCl) lysis (12). The leukocytes were then washed in Gey's medium and resuspended in Gey's medium to contain 3.3 x 106 total cells/ml. The cell suspension was placed in the upper
489
compartment of the chemotaxis chamber which was separated from the stimulant with a 5-,um nitrocellulose filter (Millipore Corp., New Bedford, Mass.). After incubation for 3 h in humidified air at 37 C, the filters were fixed and stained (11) and chemotaxis was quantified by counting and averaging the PMNs which migrated completely through the filter (20 high-power fields, x780). Complement activators. Endotoxic lipopolysaccharide (endotoxin) derived from Salmonella typhosa 0901 was purchased from Difco Laboratories, Detroit, Mich. Aggregated human gamma globulin (AHGG) was prepared by heating immune serum globulin (Armour Pharmaceutical Co., Pheonix, Ariz.) for 10 min at 61 C. Production of chemotactic activity. Mouse serum (0.3 ml) was incubated for 60 min at 37 C with 0.2 ml of gelatin Veronal buffer supplemented with Mg2+ and Ca2+ (GVB2+) (7) containing various concentrations of endotoxin or AHGG, or 0.2 ml of GVB2+ alone. Samples were then heat inactivated for 30 min at 56 C and spun at 500 x g for 10 min to remove aggregates. For use in the chemotaxis assay, 0.085 ml of the appropriate serum sample was added to 0.165 ml of GVB2 , pH 7.0, brought to 0.85 ml with Gey's medium, and was placed in the lower compartment of a modified Boyden chamber. Molecular sieve fractionation of mouse serum. Serum samples (3.0 ml) were applied to a Sephadex G-100 (Pharmacia Fine Chemicals, Uppsala, Sweden) column (2.5 by 50 cm) which had previously been equilibrated at 4 C with phosphate (0.02 M)-buffered (pH 7.2) isotonic saline. Fractions were collected and the absorbance at 280 nm was determined on a GCAlMcPherson double beam spectrophotometer (Alton, Mass.). Fractions were assayed for macrophage or PMN chemotactic activity.
RESULTS Production of chemotactic activity for macrophages by endotoxin and AHGG in normal mouse serum. To determine if the interaction of complement activators such as endotoxin and AHGG (5) with mouse serum produced chemotactic activity for macrophages, normal serum was incubated with various concentrations of these agents and then tested for chemotactic activity (Table 1). Doses of as little as 1.5 ,ug of endotoxin or 3.0 ,ug of AHGG produced significant amounts of chemotactic activity in both sources of normal mouse serum. Moreover, increasing the dose of either inflammatory agent produced increasing amounts of chemotactic activity. Preheating the serum (56 C for 30 min) to destroy the complement activity prevented the generation of chemotactic activity by either inflammatory agent.
Lack of production of chemotactic activity for macrophages by endotoxin and AHGG in
490
SNYDERMAN ET AL.
TABLE 1. Production of chemotactic activity by endotoxin and AHGG in normal mouse serum Chemotactic activity"
Complement activatora
C3H/HeJ
Endotoxin (.ug) 1.5 15 75 150 300
AHGG (,ug) 3 30 150 300 600
10.6 32.0 ± 43.2 ± 56.4 71.6
3.77c 1.46 2.36 0.27 3.30
25.2 0.16 42.7 ± 2.94 48.5 ± 2.19 56.8±2.14 65.0 2.19
CBA/J
22.0 ± 31.0 4 57.1 ± 52.5 64.0
3.77 2.71 3.07 6.17 5.01
6.6 0.99 23.1 ± 2.33 34.9 ± 3.94 38.9±3.42 50.8 3.46
Mouse serum (0.3 ml) was incubated (37 C for 60 min followed by 56 C for 30 min) with 0.2 ml of GVB2+ containing the indicated amount of endotoxin or AHGG or with 0.2 ml of GVB2+ alone. Samples were then centrifuged for 10 min at 500 x g and tested for chemotactic activity. b Net chemotactic activity was determined by subtracting the chemotactic activity in serum which had been incubated with GVB2+ alone (C3H/HeJ = 42.9 ± 3.14, CBA/J = 47.5 ± 1.86) from the chemotactic activity in serum which had been incubated with GVB2+ containing the indicated amounts of complement activator. The migratory response of cells to medium alone was less than 2.0. C3H/HeJ and CBA/J are serum sources. c Mean of triplicate samples expressed as macrophages per oil immersion field (x 1540) (+ standard error of mean).
C5-deficient mouse serum. To determine directly if the fifth component of complement was required for the production of chemotactic activity for macrophages in mouse serum, serum from two lines of C5-deficient mice were treated with various doses of endotoxin and AHGG (Table 2). Doses of endotoxin ranging from 1.5 to 300 gg and AHGG ranging from 3.0 to 600 gg failed to produce any chemotactic activity for macrophages. It was also noted that the background chemotactic activity in untreated C5-deficient mouse serum was substantially less than that of untreated normal mouse serum (see footnote b in Tables 1 and 2). Characterization of chemotactic activity for macrophages in mouse serum. Normal mouse serum and normal mouse serum treated with endotoxin was applied to a Sephadex G-100 column to determine the elution profile of the chemotactic activity produced by inflammatory agents as well as the nature of the chemotactic activity present in untreated se-
INFECT. IMMUN.
rum. C5-deficient serum treated with endotoxin was similarly fractionated to determine if activity not detectable in whole serum could be
unmasked by molecular sieve chromatography. Untreated normal serum contained a single peak of chemotactic activity corresponding to a molecular weight of greater than 68,000. Endotoxin-treated normal serum, in addition to the chemotactic activity present in untreated normal serum, contained a second peak of greater activity (ca. 15,000 daltons) eluting just before a cytochrome c marker (Fig. 2a and b). In contrast, endotoxin-treated serum from C5deficient mice contained neither the highmolecular-weight background chemotactic activity nor the low-molecular-weight chenmotactic factor produced upon treatment of normal serum with endotoxin (Fig. 2c). Fractions of activated normal serum (Fig. 2b) were also tested for PMN chemotactic activity, and the peak of activity of mouse PMNs coincided with the low-molecular-weight peak of TABLE 2. Lack of production of chemotactic activity by endotoxin and AHGG in C5-deficient mouse serum Complement activatorP
Chemotactic activity"
AKR/J
Endotoxin (,ug) 1.5 15 75
150 300
A/J
0.0 ± O.0c 0.5 ± 0.36 2.0 ± 1.41 0.3 ± 0.40 1.9 2.31
1.7 ± 2.0 ± 3.7 ± 1.8 ± 0.0
2.2 2.2 ± 1.4 1.5 1.2
0.0 0.0 0.4 ± 0.54 1.2 0.35 2.0 2.24 0.0 ± 0.0
2.02 2.42 2.40 0.26 0.0
AHGG (gg) 3 30 150 300 600
0.94 1.44 1.76 0.94 1.51
a Mouse serum (0.3 ml) was incubated (37 C for 60 min followed by 56 C for 30 min) with 0.2 ml of GVB2+ containing the indicated amount of endotoxin or AHGG or with 0.2 ml of GVB2+ alone. Samples were then centrifuged for 10 min at 500 x g and tested for chemotactic activity. h Net chemotactic activity was determined by subtracting the chemotactic activity in serum which had been incubated with GVB2+ alone (A/J = 7.6 ± 0.7, AKR/J = 6.93 ± 2.35) from the chemotactic activity in serum which had been incubated with GVB2+ containing the indicated amount of complement activator. The migratory response of cells to medium alone was less than 3.5. AKR/J and A/J are serum sources. cMean of triplicate samples expressed as macrophages per oil immersion field (x1540) (+ standard error of mean).
VOL. 11, 1975
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EIuOte (ml) FIG. 2. Molecular sieve fractionation of mouse serum. C5 normal (CBA/J) or C5-deficient (AKR/J) mouse serum (3.0 ml) was incubated with 3.0 mg of endotoxin contained in 0.3 ml of GVB2+ or with 0.3 ml of GVB2+ alone for 60 min at 37 C, followed by 30 min at 56 C. The samples were then centrifuged for 10 min at 500 x g, and the supernatant was applied to a G-100 Sephadex column (2.5 by 50 cm). Fractions (3.5 ml) were eluted with phosphate (0.02 M)-buffered saline (pH 7.2) with a flow rate of approximately 10 ml/h. For use in the chemotaxis assay, 0.4 ml of each fraction was brought to 0.85 ml with Gey's medium, placed in the lower compartment of a modified Boyden chamber, and tested for macrophage or PMN Volume of
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chemotactic activity for mouse macrophages. This peak of chemotactic activity for mouse PMNs has previously been shown to be attributable to C5a (13).
DISCUSSION Macrophages function to process antigens, phagocytize tissue breakdown products at sites of wounds, and act as effector cells in immunologically mediated inflammatory reactions. The mechanism of macrophage accumulation at local sites is therefore of obvious importance for a more complete understanding of host defense against trauma, infection, and neoplasia. The data presented herein demonstrates the feasibility of studying chemotaxis of peritoneal mac-
491
rophages derived from the mouse, an animal extensively used to study immune parameters of host defense. It has previously been shown that C5a, a low-molecular-weight cleavage product released upon activation of C5, is chemotactic for PMNs derived from many species (12, 21) and for human monocytes (10) as well as guinea pig (14, 16) and rabbit peritoneal macrophages (19, 22). Wissler et al. (24), however, have presented data which suggest that C5a, although chemotactic for neutrophils and eosinophils, is not chemotactic for macrophages. Nevertheless, the data presented herein shows that the chemotactic activity for macrophages, as well as for PMNs, produced in mouse serum is dependent upon the presence of the fifth component of complement. Serum from mice congenitally devoid of C5 produced no chemotactic activity for macrophages upon incubation with inflammatory agents such as endotoxin or aggregated gamma globulin. Moreover, the activity produced in activated normal mouse serum is identical in its G-100 Sephadex elution profile to that of the chemotactic activity for PMNs and macrophages present in human and guinea pig serum treated with complement activators. The activity in the latter serums has previously been shown to be attributable to C5a (15). It is interesting to note that in addition to the low-molecular-weight chemotactic factor which is presumably C5a, there is additional chemotactic activity present in normal mouse serum but not in C5-deficient serum. High-molecularweight background chemotactic activity in untreated serum of other species has been previously reported but never clearly identified (6). The nature of this heavy-molecular-weight chemotactic factor(s) is not yet known, but this activity is clearly absent in C5-deficient serum and thus C5 is required for its presence. ACKNOWLEDGMENTS This work was supported in part by Public Health Research grant 5 R01 DE 03738 from the National Institute of Dental Research and contract NOI CP 33313 from the National Cancer Institute. R. Snvderman is a Howard Hughes Medical Investigator. LITERATURE CITED 1. Altman, L. C., and H. Kirchner. 1972. The production of a monocyte chemotactic factor by agammaglobulinemic chicken spleen cells. J. Immunol. 109:1149-1151. 2. Boyden, S. 1962. The chemotactic effect of mixtures of antibody and antigen on polvmorphonuclear leukocytes. J. Exp. Med. 115:453-466. 3. Cinader, B., S. Dubiski, and A. C. Wardlaw. 1964. Distribution, inheritance and properties of an antigen, MuBl, and its relation to hemolytic complement. J. Exp. Med. 120:897-924.
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4. Erickson, R. P., D. K. Tachibana, L. A. Herzenberg, and L. T. Rosenberg. 1964. A single gene controlling hemolytic complement and a serum antigen in the mouse. J.
5.
6. 7.
8.
9.
10.
11.
12.
Immunol. 92:611-615. Gewurz, H., H. S. Shin, and S. E. Mergenhagen. 1968. Interactions of the complement system with endotoxic lipopolysaccharides. Consumption of each of the six terminal complement components. J. Exp. Med. 128: 1049-1057. Hausman, M. S., R. Snyderman, and S. E. Mergenhagen. 1972. Humoral mediators of chemotaxis of mononuclear leukocytes. J. Infect. Disease. 125:595-602. Mayer, M. M. 1961. Complement and complement fixation, p. 133-240. In E. A. Kabat and M. M. Mayer (ed.), Experimental immunochemistry, 2nd ed. Charles C Thomas, Springfield, Ill. Nilsson, U. R., and H. J. Muller-Eberhard. 1967. Deficiency of the fifth component of complement in mice with an inherited complement defect. J. Exp. Med. 125: 1-16. Snyderman, R., L. C. Altman, A. Frankel, and R. M. Blaese. 1973. Defective mononuclear leukocyte chemotaxis, a previously unrecognized immune dysfunction: studies in a patient with chronic mococutaneous candidiasis. Ann. Int. Med. 78:509-513. Snyderman, R., L. C. Altman, M. S. Hausman, and S. E. Mergenhagen. 1972. Human mononuclear leukocyte chemotaxis: a quantitative assay for mediators of humoral and cellular chemotactic factors. J. Immunol. 108:857-860. Snyderman, R., H. Gewurz, and S. E. Mergenhagen. 1968. Interactions of the complement system with endotoxic lipopolysaccharide. Generation of a factor chemotactic for polymorphonuclear leukocytes. J. Exp. Med. 128:259-275. Snyderman, R., and S. E. Mergenhagen. 1972. Characterization of polymorphonuclear leukocyte chemotactic activity in serums activated by various inflammatory agents, p. 117-132. In S. Karger (ed.), Biological activities of complement. Fifth Int. Symp. Can. Soc. Immunol., Guelph. Basal, Switzerland.
INFECT. IMMUN.
13. Snyderman, R., J. K. Phillips, and S. E. Mergenhagen. 1971. Biological activity of complement in vivo: role of
14.
15.
16.
17. 18.
19.
20. 21. 22.
23. 24.
C5 in the accumulation of polymorphonuclear leukocytes in inflammatory exudates. J. Exp. Med. 134:1131-1143. Snyderman, R., H. S. Shin, and M. S. Hausman. 1971. A chemotactic factor for mononuclear leukocytes. Proc. Soc. Exp. Biol. Med. 138:387-390. Snyderman, R., H. S. Shin, J. K. Phillips, H. Gewurz, and S. E. Mergenhagen. 1969. A neutrophil chemotactic factor derived from C5 upon interaction of guinea pig serum with endotoxin. J. Immunol. 103:413-422. Wahl, S. M., L. C. Altman, J. J. Oppenheim, and S. E. Mergenhagen. 1974. In vitro studies of a chemotactic lymphokine in the guinea pig. Int. Arch. Allergy 46:768-784. Ward, P. A. 1969. A leukotactic factor produced by sensitized lymphocytes. Science 163:1079-1081. Ward, P. A. 1971. Leukotactic factors in health and disease. Am. J. Pathol. 64:521-530. Ward, P. A., and J. L. Berenberg. 1974. Defective regulation of inflammatory mediators in Hodgkin's disease: super normal levels of chemotactic factor inactivator. N. Engl. J. Med. 290:75-80. Ward, P. A., C. G. Cochrane, and H. J. Muller-Eberhard. 1965. The role of serum complement in chemotaxis of leukocytes in vitro. J. Exp. Med. 122:327-346. Ward, P. A., and L. J. Newman. 1969. A neutrophil chemotactic factor from human C5. J. Immunol. 102:93-99. Ward, P. A., H. G. Remold, and J. R. David. 1970. The production by antigen-stimulated lymphocytes of a leukotactic factor distinct from migration inhibition factor. Cell. Immunol. 1:162-174. Wilkinson, P. C. 1974. Clinical disorders of chemotactic function, p. 140-148. In Chemotaxis and inflammation. Churchill-Livingstone, Edinburgh. Wissler, J. H., V. J. Stecher, and E. Sorkin. 1972. Biochemistry and biology of a leukotactic binary serum peptide system related to anaphylatoxin. Int. Arch. Allergy Appl. Immunol. 42:722-747.
Vol. 11. No. 3 Printed in U.S.A.
Quantification of Mouse Macrophage Chemotaxis in Vitro: Role of C5 for the Production of Chemotactic Activity RALPH SNYDERMAN,* MARILYN C. PIKE, DEAN McCARLEY, AND LAWRENCE LANG Division of Rheumatic and Genetic Disease, Departments of Medicine and Immunology, Duke University Medical Center, Durham, North Carolina 27710 Received for publication 27 November 1974
Delineation of the mechanisms of macrophage accumulation at local tissue sites will further our understanding of immunologically mediated host resistance to infectious and neoplastic diseases. Since mice are frequently used for the study of immune function, we developed a method for the quantification of mouse macrophage chemotaxis in vitro. By this method it was found that the fifth component of complement is necessary for the production of chemotactic activity in mouse serum by inflammatory agents such as endotoxin or aggregated gamma globulin. The majority of macrophage chemotactic activity produced by these agents in mouse serum can be attributed to a low-molecular-weight (ca. 15,000) chemotactic factor. The data suggest that this factor is the biologically active cleavage product of the fifth component of complement, C5a.
Accumulation of macrophages at local tissue sites is an important event in wound healing and in immunologically mediated host defense. Methods for the quantification of macrophage migration can therefore provide valuable tools for the study of factors involved in host resistance to infectious and neoplastic diseases. The availability of quantitative methods for the study of leukocyte chemotaxis in vitro (2, 10, 14, 15, 18) has resulted in the characterization of several chemotactic factors as well as the delineation of abnormalities of leukotaxis in certain human disease states (9, 18, 19, 22). Although macrophage chemotaxis assays have been developed for a number of animal species (1, 10, 14, 20), no method has been described to measure mouse macrophage migration in vitro. Since many studies of immunological parameters of host resistance are performed in mice, we developed a quantitative, reproducible assay for mouse macrophage chemotaxis and characterized factors in mouse serum which are chemotactic for macrophages in vitro.
Macrophages for the chemotaxis assay.
C3H/HeJ mice were injected intraperitoneally with
MATERIALS AND METHODS Mice. Male mice of the lines C3H/HeJ and CBA/J (C5 normal) and AKR/J and A/J (C5 deficient) (3, 4, 8) were purchased from Jackson Laboratories, Bar Harbor, Me. Mouse Serum. Blood from normal and C5-deficient mice was obtained from the retro-orbital plexus or by cardiac puncture and allowed to clot at room temperature for 30 min. The serum was removed after centrifugation and stored at -70 C until use. 488
2.0 ml of 10% proteose peptone (Difco Laboratories, Detroit, Mich.) in water 3 or 4 days before sacrifice. Mice were sacrificed by CO, asphyxiation and the peritoneal cavities were exposed by abdominal incision. The cavities were then washed with approximately 7.5 ml of Gey's balanced salt solution, pH 7.0 (Flow Laboratories, Rockville, Md.), containing 2% bovine serum albumin, 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (Calbiochem, La Jolla, Calif.) (Gey's medium), and. 10 U of heparin/ml. The peritoneal exudates from several mice were pooled and centrifuged (300 x g) at 4 C for 10 min. Approximately 8 x 10" leukocytes were obtained per mouse, and of these about 80% were macrophages and 20% were lymphocytes. The cells were washed one time in Gey's medium, and resuspended to contain 1.5 x 106 viable macrophages/ml for use in the chemotaxis assay. Viability was determined by trypan blue exclusion and generally at least 97% of the cells were viable when collected by these methods. Macrophages chemotaxis assay. Various materials were tested for chemotactic activity in vitro using a modification of methods described previously (14). In brief, the chemotactic stimulant under study was placed in the lower compartment of a modified Boyden chamber (Fig. 1) and separated by a 5-tsm polycarbonate (Nuclepore) filter (Wallabs, San Rafael, Calif.) from the upper compartment containing the cell suspension. The upper compartment of the chemotaxis chamber (Duke University Surgical Instrument Shop, Durham, N. C.) holds 0.4 ml of the cell suspension, while the lower compartment holds 0.85 ml of the chemotactic or control stimulant. All assays were performed in triplicate and the chambers
VOL. 11, 1975
MOUSE MACROPHAGE CHEMOTAXIS IN VITRO
Upper Chomber
1F~~~~~~~~~ Ite Lower
Chamber
FiG. 1. Chemotaxis chamber. A standardized cell suspension (0.4 ml) is placed in the upper compartment of the chamber and is separated from the chemotactic stimulant (0.85 ml) or medium alone in the lower compartment by a 5-,gm pore size polycarbonate or nitrocellulose filter. containing macrophages and stimulants were incubated for 4 h in humidified air at 37 C. After incubation, the chambers were emptied and the filters were removed; then fixed in ethanol for 15 s, stained for 6 min in Mayer's hematoxylin, 1 min in acid alcohol, and 1 min in bluing solution (11). Chemotaxis was quantified by counting and averaging the macrophages in 20 oil immersion fields, (x 1,540) that migrated completely through the filter. PMN chemotaxis assay. Mouse polymorphonuclear leukocytes (PMN) were isolated as described previously (13). Briefly, C3H/HeJ mice were bled by cardiac puncture into syringes containing approximately 20 U of heparin per ml of blood. The blood was mixed with an equal volume of 3% dextran (Dextran T 250, Pharmacia Fine Chemicals, Uppsala, Sweden) in normal saline. The erythrocytes were allowed to sediment for 30 min at room temperature and the leukocyte-rich plasma was withdrawn. The plasma was then centrifuged at 300 x g for 10 min and the contaminating erythrocytes were removed by rapid (20 s) hypotonic (0.2% NaCl) lysis (12). The leukocytes were then washed in Gey's medium and resuspended in Gey's medium to contain 3.3 x 106 total cells/ml. The cell suspension was placed in the upper
489
compartment of the chemotaxis chamber which was separated from the stimulant with a 5-,um nitrocellulose filter (Millipore Corp., New Bedford, Mass.). After incubation for 3 h in humidified air at 37 C, the filters were fixed and stained (11) and chemotaxis was quantified by counting and averaging the PMNs which migrated completely through the filter (20 high-power fields, x780). Complement activators. Endotoxic lipopolysaccharide (endotoxin) derived from Salmonella typhosa 0901 was purchased from Difco Laboratories, Detroit, Mich. Aggregated human gamma globulin (AHGG) was prepared by heating immune serum globulin (Armour Pharmaceutical Co., Pheonix, Ariz.) for 10 min at 61 C. Production of chemotactic activity. Mouse serum (0.3 ml) was incubated for 60 min at 37 C with 0.2 ml of gelatin Veronal buffer supplemented with Mg2+ and Ca2+ (GVB2+) (7) containing various concentrations of endotoxin or AHGG, or 0.2 ml of GVB2+ alone. Samples were then heat inactivated for 30 min at 56 C and spun at 500 x g for 10 min to remove aggregates. For use in the chemotaxis assay, 0.085 ml of the appropriate serum sample was added to 0.165 ml of GVB2 , pH 7.0, brought to 0.85 ml with Gey's medium, and was placed in the lower compartment of a modified Boyden chamber. Molecular sieve fractionation of mouse serum. Serum samples (3.0 ml) were applied to a Sephadex G-100 (Pharmacia Fine Chemicals, Uppsala, Sweden) column (2.5 by 50 cm) which had previously been equilibrated at 4 C with phosphate (0.02 M)-buffered (pH 7.2) isotonic saline. Fractions were collected and the absorbance at 280 nm was determined on a GCAlMcPherson double beam spectrophotometer (Alton, Mass.). Fractions were assayed for macrophage or PMN chemotactic activity.
RESULTS Production of chemotactic activity for macrophages by endotoxin and AHGG in normal mouse serum. To determine if the interaction of complement activators such as endotoxin and AHGG (5) with mouse serum produced chemotactic activity for macrophages, normal serum was incubated with various concentrations of these agents and then tested for chemotactic activity (Table 1). Doses of as little as 1.5 ,ug of endotoxin or 3.0 ,ug of AHGG produced significant amounts of chemotactic activity in both sources of normal mouse serum. Moreover, increasing the dose of either inflammatory agent produced increasing amounts of chemotactic activity. Preheating the serum (56 C for 30 min) to destroy the complement activity prevented the generation of chemotactic activity by either inflammatory agent.
Lack of production of chemotactic activity for macrophages by endotoxin and AHGG in
490
SNYDERMAN ET AL.
TABLE 1. Production of chemotactic activity by endotoxin and AHGG in normal mouse serum Chemotactic activity"
Complement activatora
C3H/HeJ
Endotoxin (.ug) 1.5 15 75 150 300
AHGG (,ug) 3 30 150 300 600
10.6 32.0 ± 43.2 ± 56.4 71.6
3.77c 1.46 2.36 0.27 3.30
25.2 0.16 42.7 ± 2.94 48.5 ± 2.19 56.8±2.14 65.0 2.19
CBA/J
22.0 ± 31.0 4 57.1 ± 52.5 64.0
3.77 2.71 3.07 6.17 5.01
6.6 0.99 23.1 ± 2.33 34.9 ± 3.94 38.9±3.42 50.8 3.46
Mouse serum (0.3 ml) was incubated (37 C for 60 min followed by 56 C for 30 min) with 0.2 ml of GVB2+ containing the indicated amount of endotoxin or AHGG or with 0.2 ml of GVB2+ alone. Samples were then centrifuged for 10 min at 500 x g and tested for chemotactic activity. b Net chemotactic activity was determined by subtracting the chemotactic activity in serum which had been incubated with GVB2+ alone (C3H/HeJ = 42.9 ± 3.14, CBA/J = 47.5 ± 1.86) from the chemotactic activity in serum which had been incubated with GVB2+ containing the indicated amounts of complement activator. The migratory response of cells to medium alone was less than 2.0. C3H/HeJ and CBA/J are serum sources. c Mean of triplicate samples expressed as macrophages per oil immersion field (x 1540) (+ standard error of mean).
C5-deficient mouse serum. To determine directly if the fifth component of complement was required for the production of chemotactic activity for macrophages in mouse serum, serum from two lines of C5-deficient mice were treated with various doses of endotoxin and AHGG (Table 2). Doses of endotoxin ranging from 1.5 to 300 gg and AHGG ranging from 3.0 to 600 gg failed to produce any chemotactic activity for macrophages. It was also noted that the background chemotactic activity in untreated C5-deficient mouse serum was substantially less than that of untreated normal mouse serum (see footnote b in Tables 1 and 2). Characterization of chemotactic activity for macrophages in mouse serum. Normal mouse serum and normal mouse serum treated with endotoxin was applied to a Sephadex G-100 column to determine the elution profile of the chemotactic activity produced by inflammatory agents as well as the nature of the chemotactic activity present in untreated se-
INFECT. IMMUN.
rum. C5-deficient serum treated with endotoxin was similarly fractionated to determine if activity not detectable in whole serum could be
unmasked by molecular sieve chromatography. Untreated normal serum contained a single peak of chemotactic activity corresponding to a molecular weight of greater than 68,000. Endotoxin-treated normal serum, in addition to the chemotactic activity present in untreated normal serum, contained a second peak of greater activity (ca. 15,000 daltons) eluting just before a cytochrome c marker (Fig. 2a and b). In contrast, endotoxin-treated serum from C5deficient mice contained neither the highmolecular-weight background chemotactic activity nor the low-molecular-weight chenmotactic factor produced upon treatment of normal serum with endotoxin (Fig. 2c). Fractions of activated normal serum (Fig. 2b) were also tested for PMN chemotactic activity, and the peak of activity of mouse PMNs coincided with the low-molecular-weight peak of TABLE 2. Lack of production of chemotactic activity by endotoxin and AHGG in C5-deficient mouse serum Complement activatorP
Chemotactic activity"
AKR/J
Endotoxin (,ug) 1.5 15 75
150 300
A/J
0.0 ± O.0c 0.5 ± 0.36 2.0 ± 1.41 0.3 ± 0.40 1.9 2.31
1.7 ± 2.0 ± 3.7 ± 1.8 ± 0.0
2.2 2.2 ± 1.4 1.5 1.2
0.0 0.0 0.4 ± 0.54 1.2 0.35 2.0 2.24 0.0 ± 0.0
2.02 2.42 2.40 0.26 0.0
AHGG (gg) 3 30 150 300 600
0.94 1.44 1.76 0.94 1.51
a Mouse serum (0.3 ml) was incubated (37 C for 60 min followed by 56 C for 30 min) with 0.2 ml of GVB2+ containing the indicated amount of endotoxin or AHGG or with 0.2 ml of GVB2+ alone. Samples were then centrifuged for 10 min at 500 x g and tested for chemotactic activity. h Net chemotactic activity was determined by subtracting the chemotactic activity in serum which had been incubated with GVB2+ alone (A/J = 7.6 ± 0.7, AKR/J = 6.93 ± 2.35) from the chemotactic activity in serum which had been incubated with GVB2+ containing the indicated amount of complement activator. The migratory response of cells to medium alone was less than 3.5. AKR/J and A/J are serum sources. cMean of triplicate samples expressed as macrophages per oil immersion field (x1540) (+ standard error of mean).
VOL. 11, 1975
MOUSE MACROPHAGE CHEMOTAXIS IN VITRO is. Q)
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EIuOte (ml) FIG. 2. Molecular sieve fractionation of mouse serum. C5 normal (CBA/J) or C5-deficient (AKR/J) mouse serum (3.0 ml) was incubated with 3.0 mg of endotoxin contained in 0.3 ml of GVB2+ or with 0.3 ml of GVB2+ alone for 60 min at 37 C, followed by 30 min at 56 C. The samples were then centrifuged for 10 min at 500 x g, and the supernatant was applied to a G-100 Sephadex column (2.5 by 50 cm). Fractions (3.5 ml) were eluted with phosphate (0.02 M)-buffered saline (pH 7.2) with a flow rate of approximately 10 ml/h. For use in the chemotaxis assay, 0.4 ml of each fraction was brought to 0.85 ml with Gey's medium, placed in the lower compartment of a modified Boyden chamber, and tested for macrophage or PMN Volume of
chemotactic activity: (A) normal
serum
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chemotactic activity for mouse macrophages. This peak of chemotactic activity for mouse PMNs has previously been shown to be attributable to C5a (13).
DISCUSSION Macrophages function to process antigens, phagocytize tissue breakdown products at sites of wounds, and act as effector cells in immunologically mediated inflammatory reactions. The mechanism of macrophage accumulation at local sites is therefore of obvious importance for a more complete understanding of host defense against trauma, infection, and neoplasia. The data presented herein demonstrates the feasibility of studying chemotaxis of peritoneal mac-
491
rophages derived from the mouse, an animal extensively used to study immune parameters of host defense. It has previously been shown that C5a, a low-molecular-weight cleavage product released upon activation of C5, is chemotactic for PMNs derived from many species (12, 21) and for human monocytes (10) as well as guinea pig (14, 16) and rabbit peritoneal macrophages (19, 22). Wissler et al. (24), however, have presented data which suggest that C5a, although chemotactic for neutrophils and eosinophils, is not chemotactic for macrophages. Nevertheless, the data presented herein shows that the chemotactic activity for macrophages, as well as for PMNs, produced in mouse serum is dependent upon the presence of the fifth component of complement. Serum from mice congenitally devoid of C5 produced no chemotactic activity for macrophages upon incubation with inflammatory agents such as endotoxin or aggregated gamma globulin. Moreover, the activity produced in activated normal mouse serum is identical in its G-100 Sephadex elution profile to that of the chemotactic activity for PMNs and macrophages present in human and guinea pig serum treated with complement activators. The activity in the latter serums has previously been shown to be attributable to C5a (15). It is interesting to note that in addition to the low-molecular-weight chemotactic factor which is presumably C5a, there is additional chemotactic activity present in normal mouse serum but not in C5-deficient serum. High-molecularweight background chemotactic activity in untreated serum of other species has been previously reported but never clearly identified (6). The nature of this heavy-molecular-weight chemotactic factor(s) is not yet known, but this activity is clearly absent in C5-deficient serum and thus C5 is required for its presence. ACKNOWLEDGMENTS This work was supported in part by Public Health Research grant 5 R01 DE 03738 from the National Institute of Dental Research and contract NOI CP 33313 from the National Cancer Institute. R. Snvderman is a Howard Hughes Medical Investigator. LITERATURE CITED 1. Altman, L. C., and H. Kirchner. 1972. The production of a monocyte chemotactic factor by agammaglobulinemic chicken spleen cells. J. Immunol. 109:1149-1151. 2. Boyden, S. 1962. The chemotactic effect of mixtures of antibody and antigen on polvmorphonuclear leukocytes. J. Exp. Med. 115:453-466. 3. Cinader, B., S. Dubiski, and A. C. Wardlaw. 1964. Distribution, inheritance and properties of an antigen, MuBl, and its relation to hemolytic complement. J. Exp. Med. 120:897-924.
492
SNYDERMAN ET AL.
4. Erickson, R. P., D. K. Tachibana, L. A. Herzenberg, and L. T. Rosenberg. 1964. A single gene controlling hemolytic complement and a serum antigen in the mouse. J.
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Immunol. 92:611-615. Gewurz, H., H. S. Shin, and S. E. Mergenhagen. 1968. Interactions of the complement system with endotoxic lipopolysaccharides. Consumption of each of the six terminal complement components. J. Exp. Med. 128: 1049-1057. Hausman, M. S., R. Snyderman, and S. E. Mergenhagen. 1972. Humoral mediators of chemotaxis of mononuclear leukocytes. J. Infect. Disease. 125:595-602. Mayer, M. M. 1961. Complement and complement fixation, p. 133-240. In E. A. Kabat and M. M. Mayer (ed.), Experimental immunochemistry, 2nd ed. Charles C Thomas, Springfield, Ill. Nilsson, U. R., and H. J. Muller-Eberhard. 1967. Deficiency of the fifth component of complement in mice with an inherited complement defect. J. Exp. Med. 125: 1-16. Snyderman, R., L. C. Altman, A. Frankel, and R. M. Blaese. 1973. Defective mononuclear leukocyte chemotaxis, a previously unrecognized immune dysfunction: studies in a patient with chronic mococutaneous candidiasis. Ann. Int. Med. 78:509-513. Snyderman, R., L. C. Altman, M. S. Hausman, and S. E. Mergenhagen. 1972. Human mononuclear leukocyte chemotaxis: a quantitative assay for mediators of humoral and cellular chemotactic factors. J. Immunol. 108:857-860. Snyderman, R., H. Gewurz, and S. E. Mergenhagen. 1968. Interactions of the complement system with endotoxic lipopolysaccharide. Generation of a factor chemotactic for polymorphonuclear leukocytes. J. Exp. Med. 128:259-275. Snyderman, R., and S. E. Mergenhagen. 1972. Characterization of polymorphonuclear leukocyte chemotactic activity in serums activated by various inflammatory agents, p. 117-132. In S. Karger (ed.), Biological activities of complement. Fifth Int. Symp. Can. Soc. Immunol., Guelph. Basal, Switzerland.
INFECT. IMMUN.
13. Snyderman, R., J. K. Phillips, and S. E. Mergenhagen. 1971. Biological activity of complement in vivo: role of
14.
15.
16.
17. 18.
19.
20. 21. 22.
23. 24.
C5 in the accumulation of polymorphonuclear leukocytes in inflammatory exudates. J. Exp. Med. 134:1131-1143. Snyderman, R., H. S. Shin, and M. S. Hausman. 1971. A chemotactic factor for mononuclear leukocytes. Proc. Soc. Exp. Biol. Med. 138:387-390. Snyderman, R., H. S. Shin, J. K. Phillips, H. Gewurz, and S. E. Mergenhagen. 1969. A neutrophil chemotactic factor derived from C5 upon interaction of guinea pig serum with endotoxin. J. Immunol. 103:413-422. Wahl, S. M., L. C. Altman, J. J. Oppenheim, and S. E. Mergenhagen. 1974. In vitro studies of a chemotactic lymphokine in the guinea pig. Int. Arch. Allergy 46:768-784. Ward, P. A. 1969. A leukotactic factor produced by sensitized lymphocytes. Science 163:1079-1081. Ward, P. A. 1971. Leukotactic factors in health and disease. Am. J. Pathol. 64:521-530. Ward, P. A., and J. L. Berenberg. 1974. Defective regulation of inflammatory mediators in Hodgkin's disease: super normal levels of chemotactic factor inactivator. N. Engl. J. Med. 290:75-80. Ward, P. A., C. G. Cochrane, and H. J. Muller-Eberhard. 1965. The role of serum complement in chemotaxis of leukocytes in vitro. J. Exp. Med. 122:327-346. Ward, P. A., and L. J. Newman. 1969. A neutrophil chemotactic factor from human C5. J. Immunol. 102:93-99. Ward, P. A., H. G. Remold, and J. R. David. 1970. The production by antigen-stimulated lymphocytes of a leukotactic factor distinct from migration inhibition factor. Cell. Immunol. 1:162-174. Wilkinson, P. C. 1974. Clinical disorders of chemotactic function, p. 140-148. In Chemotaxis and inflammation. Churchill-Livingstone, Edinburgh. Wissler, J. H., V. J. Stecher, and E. Sorkin. 1972. Biochemistry and biology of a leukotactic binary serum peptide system related to anaphylatoxin. Int. Arch. Allergy Appl. Immunol. 42:722-747.
