Vol. 18, No. 1 Printed in U.S.A.
INFECTION AND IMMUNITY, Oct. 1977, p. 102-109 Copyright © 1977 American Society for Microbiology
Canine Migration Inhibitory Factor: Effect of Corynebacterium parvum Administration MAYA S. PINEIRO,1 CHARLES A. BOWLES,`* ERNEST C. CUTCHINS,2 AND MALCOLM I. BULLt
Department of Microbiology, Hazleton Laboratories America, Inc., Vienna, Virginia 221801; Department of Biology, Catholic University of America, Washington, D.C. 200642; and Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland 20014
Received for publication 5 April 1977
Peripheral blood lymphocytes from dogs sensitized to streptolysin 0 (SLO) were assayed for migration inhibitory factor (MIF) production by the indirect MIF test, using guinea pig peritoneal exudate cells as the source of macrophages. A specific direct correlation was established between the degree of inhibition of migration and the concentration of SLO-stimulated supernatants from lymphocyte cultures (SLO-S) of untreated normal dogs. Undiluted SLO-S inhibited migration by 66.8%, whereas a dilution of 1:64 elicited a 3% inhibition. In parallel tests, purified protein derivative stimulation of lymphocytes from BCG-vaccinated dogs produced 92.6% inhibition. The effect of Corynebacterium parvum on SLO-specific MIF production was evaluated in three groups of dogs administered a single intramuscular injection of C. parvum at 5 or 50 mg/m2 or 50 mg/mi in suspension with 10 mg of methylprednisolone. Inhibition of migration of macrophages exposed to a 1:4 dilution of SLO-S from dogs inoculated with C. parvum (5 mg/in2) was 33% greater (mean inhibition, 75%) than the same SLOS dilution from uninoculated normal dogs (mean inhibition, 42%) (P < 0.0002). Similarly, lymphocytes from dogs administered 50 mg/m2 caused an enhancement of migration inhibition, with a mean increase of 26% over controls (P < 0.002), whereas a dose of 50 mg/m2 with methylprednisolone produced a 16% increase in migration inhibition (P < 0.05). The administration of C. parvum resulted in a three- to fourfold increase in the SLO-S dilution, which would reduce migration by 20% (MIF titer). This increase peaked between days 20 and 30 and lasted over 50 days post-C. parvum inoculation. These findings indicate that C. parvum specifically increases MIF production by canine lymphocytes in a linear correlation with SLO concentration and suggest its use as a stimulant of canine immunity. Migration inhibitory factor (MIF) is one of a class of lymphokines released by sensitized thymus-derived (T) and bone marrow-derived (B) lymphocytes upon in vitro exposure to specific antigen. These mediators affect the behavior of inflammatory cells, induce mitogenic responses in lymphocytes, participate in cytotoxicity for target cells, promote chemotaxis, aggregate leukocytes, and, in the case of MIF, inhibit the migration of macrophages (6). MIF production is considered an in vitro correlate of cell-mediated immunity (CMI), and the capacity of lymphocytes to produce MIF is used widely as a measure of delayed-type hypersensitivity (11). CMI is important in defense against infection with most viruses and in host resistance to tumors, lymphocytes and macrophages being the effector cells in the recognition and destruction process (12, 19, 29). The role of macrophages in t Present address: 691 Myrtle Lane, Grand Junction, CO 81501.
CMI is intimately linked to and may be directed by sensitized lymphocytes (1). Lymphocyte-released mediators induce a variety of changes in the macrophage periphery and result in increased phagocytic, spreading, and adhesion capacities (28). These changes are believed necessary for the expression of an effective cell-mediated immune response. Experimental tumor systems in rodents have clearly established the role of CMI in host-tumor interactions (27). Nonspecific stimulation of the immune system has been attempted with adjuvants such as BCG and MER (methanol extraction residue of BCG) but has met with limited success due to the variability of strains, number of viable organisms used, and the requirement for repeated regional administration (24-26). Nonspecific immune stimulation with Corynebacterium parvum is now possible with a killed vaccine of standardized potency. C. parvum is a strong immunopotentiator, stimulating antibody 102
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synthesis and enhancing the immune system's reaction to bacteria and viruses (17). Studies conducted in mice, rats, guinea pigs, and rabbits demonstrate a beneficial influence by C. parvum on the host in a variety of experimental neoplastic systems. Enhanced survival, decreased rate of tumor growth and metastatic dissemination, resistance to chemical carcinogens, and immunity to tumor rechallenge are some of the consequences of C. parvum administration (14, 31, 35). This report describes the effect of intramuscular administration of C. parvum on the CMI response of dogs, using the MIF assay as an indicator of lymphocyte-directed macrophage function. As dogs express naturally acquired sensitivity to streptolysin 0 (SLO), the capacity of lymphocytes to produce MIF after challenge in vitro with SLO antigen was tested to assess the action of C. parvum on their CMI. (This investigation was in partial fulfillment, by M.S.P., for the requirements for the Ph.D. degree in the Department of Biology, Catholic University of America, Washington, D.C.)
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incomplete Freund adjuvant (Difco Laboratories, Detroit, Mich.). Macrophage migration inhibition assay. A modification of the indirect guinea pig macrophage migration inhibition assay (16) was used. Peritoneal exudate cells were obtained after intraperitoneal inoculation of guinea pigs with 15 ml of emulsified incomplete Freund adjuvant (Difco Laboratories). After 72 h the peritoneal cavity was washed with Waymouth medium containing 2 U of heparin per ml. The collected wash was centrifuged at 100 x g for 10 min, and the pelleted cells were washed in Waymouth medium, again pelleted, and finally suspended at 2 x 107 cells per ml in Waymouth medium. This preparation contained greater than 75% macrophages. Siliconized (Siliclad, Clay-Adams, Parsippany, N.J.) capillary tubes (25 Al; Richard-Allan Medical Industries, Evanston, Ill.) were filled with the cell suspension and flamesealed at one end. The tubes were centrifuged at 200 x g for 10 min and cut at the cell-fluid interface. Duplicate tubes were placed in Mackaness chambers (Berton Plastics, Hackensack, N.J.) and mounted with silicone grease. Triplicate chambers were used for each experimental point, and migration values were averaged. The chambers were assembled by fixing 22-mm, siiconized, round cover glasses on a polycarbonate ring. The supernatants from the lymphocyte cultures MATERIALS AND METHODS were diluted in Waymouth medium and injected into Animals. Adult, male foxhounds housed in indoor the chambers. Each chamber was completely filed, runs and fed standard ration with water available ad the openings were grease-sealed, and the chambers libitum were used throughout the studies. Hartley were incubated at 37°C in 5% CO2 in air for 24 h. strain adult guinea pigs weighing 600 to 800 g (Charles The area of macrophage migration (length by width) River Breeding Laboratories, Wilmington, Mass.) was measured by microscopic reading through a miwere used as a source of peritoneal macrophages. crometer eyepiece (Leitz Limited, Wetzlar, Germany). C. parvum. Dogs were inoculated intramuscularly The percentage of inhibition of migration was calcuin the left hind leg with a single injection of Formol- lated as follows: percent inhibition = 100 - (average killed suspension of C. parvum, batch PX425, 7 mg area of migration in experimental supernatant x (dry weight)/ml (Weilcome Research Laboratory, 100)/(average area of migration in control supernaBeckenham, Kent, England). The dogs were divided tant). Migration inhibition of 20% or greater was coninto three groups (two animals per group), each re- sidered significant (10). Results were evaluated by the ceiving a different concentration of C. parvum. Groups two-tailed Student t test. 1 and 2 were inoculated with 5-mg/ml' (body surface Lymphocyte separation and SLO-specific MIF area) and 50-mg/m2 doses, respectively. Group 3 was production. Lymphocytes were isolated from hepainoculated with 50 mg of C. parvum per m2 mixed rinized canine peripheral blood by centrifugation on with 10 mg of methylprednisolone (Depomedrol, Up- a Ficoll-Hypaque gradient (LSM, Litton Bionetics, john Co., Kalamazoo, Mich.) to avoid the inflamma- Kensington, Md.) by the method of Boyum (7). The tion caused by the high concentration. All doses used lymphocyte layer at the interface was collected and washed twice in Waymouth medium MB 752/1 with were in a total saline inoculum of 1 ml. BCG vaccine. A suspension of 108 viable units of HEPES buffer (N-2-hydroxyethyl piperazine-N'-2Tice BCG vaccine per ml, lot 1L74C (S) 18 (University ethanesulfonic acid; Grand Island Biological Co., of Illinois, Chicago, Ill.), was used to inoculate (0.1 Grand Island, N.Y.). The final cell pellet was susml/site) intradermally five sites in the back of one pended to 107 cells per ml in Waymouth medium with dog. Peripheral blood lymphocytes were incubated for 10ug of gentamicin sulfate per ml (Gentocin, Schering 24 h with 160 ,ug of purified protein derivative (PPD) Veterinary, Kenilworth, N.J.) and 100 yg of disodium per ml (Connaught Laboratories, Toronto, Ont., Can- carbenicillin per ml (Geopen, Roerig, Pfizer Inc., N.Y.). Each preparation contained greater than 90% lymphoada). Skin test. The BCG-vaccinated dog was inoculated cytes. One-milliliter samples of purified lymphocytes intradermally with 250 tuberculin units (5 ,ug in 0.1 at 10' cells per ml were dispensed in 12- by 75-mm ml) of PPD (Mantoux batch CT68, Connaught Labo- culture tubes. Three-tenth milliliter of lyophilized ratories) in the shaved inguinal region of the right SLO reagent (Difco Laboratories; Bacto 0482 stock hind leg. The inoculation site was observed daily for reconstituted to 25 ml with Waymouth medium) was added to each culture tube and incubated for 24 h at duration and redness. Freund adjuvant. The back of one dog was inoc- 37°C in a 5% C02-air atmosphere. Four control superulated intradermally at five sites, at 0.1 ml/site, with natants were examined in 10 standardization experi-
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ments previous to C. parvum treatment. Controls were designed to eliminate underlying reactivity to SLO in the guinea pig cells and dog lymphocytes. Migration of guinea pig exudate cells incubated with SLO alone (I) was compared to the migration area in the presence of medium without the antigen (II). Migration in chambers containing supernatants from normal dog lymphocyte cultures incubated with an equal volume of antigen-free medium (III) and chambers with supernatants from lymphocytes cultured without SLO to which SLO was added after the incubation period (IV) were also compared. As values for migration areas in the four controls were not significantly different from one another using Student's t test, IV was chosen for performing the analysis of percent migration inhibition with regard to SLO and is hereafter referred to as control supernatant. After incubation, supernatants for use in tests of MIF activity were harvested after centrifugation at 200 x g for 10 min. MIF assays were performed before C. parvum injections (10 tests) and twice a week after C. parvum inoculation from day 2 through day 50.
RESULTS
Specificity of macrophage migration. To verify the specificity of the macrophage migration inhibition elicited by a recall antigen in our assay, the known BCG-PPD stimulation system was tested. Lymphocytes from a BCG-inoculated dog were incubated with 160 ,ug of PPD per ml, and the resulting supernatants were evaluated for MIF production by using guinea pig exudate cells. Before lymphocyte procurement, the BCG-vaccinated dog was demonstrated by skin tests to be PPD sensitive. Induration and erythematous skin reactions to PPD were evident at 24 h and were maximal at 72 h after inoculation. The diameter of induration ranged
INFECT. IMMUN.
from 1.5 to 4 cm at the five sites, with a mean of 2.5 cm. Table 1 presents results of MIF assays of undiluted supernatants from PPD-stimulated lymphocytes and the various controls assayed. In five tests the mean migration inhibition produced by lymphocyte supernatants from the BCG-vaccinated dog stimulated with PPD (supernatant 1) was found to be 91.8% with a mean area of migration of 0.9 x 103 ,um2 and a range from 0.6 x 103 to 1.8 x 103 ,m2. This is compared to a mean area of 11.0 x 10 Am2 with a range of 10 x 103 to 15 x 103, m2 for the uninoculated control culture consisting of lymphocytes from an uninoculated dog incubated with an equal dose of PPD (supernatant 2). The specificity of the macrophage migration was further determined by assaying lymphocytes from an uninoculated dog incubated without antigen to which PPD was added after the period of incubation (supernatant 3). When compared to this control, supernatant 1 inhibited migration by 92.6%. Various other control supernatants were evaluated for nonspecific suppression of migration. Macrophages were cultured with: lymphocyte supernatants from a dog inoculated with incomplete Freund adjuvant incubated with PPD (supernatant 4); with PPD in the absence of lymphocyte supernatant (supernatant 5); with lymphocyte supernatants from uninoculated dogs incubated in antigen-free medium (supernatant 6); and macrophages alone (supernatant 7). In all cases (supernatants 2 to 7) migration values were not significantly different from one another using the 95% confidence limit of the two-tailed Student t test. When compared with supernatant
TABLE 1. Guinea pig macrophage migration with undiluted lymphocyte supernatants from control and BCG-vaccinated dogs Supematant source, dog treatMean migration Migration range Migration inhiStimulating antigen (Mg/ml) SiuaigatgnQgm) ment areaa (Am' x 10:') bition (%) (AMm, x 10'.) 1, BCG vaccinated PPD (160) 0.9 ± 0.2" 0.6- 1.8 91.8 (2) 92.6 (3) 2, Uninoculated PPD (160) 11.0 ± 1.3d 10.0-15.0 (2) 9.1 (3) 3, Uninoculated None (PPD added) 12.1 ± 0.6 10.3-14.0 (3) 0 (2) 4, Incomplete Freund inocPPD (160) 12.4 ± 1.1 9.9-16.5 0 (3) ulated 5, None PPD (160) 12.7 ± 0.03 12.0-13.5 0 (3) 6, Uninoculated None 11.2 ± 0.9 9.1-13.5 7.4 (3) 7, None None (medium) 13.0 ± 1.7 9.1-19.1 0 (3) a ± standard error of the mean. bMigration area of supernatant 1 was significantly lower (P < 0.0002) compared to controls 2 to 7, using the two-tailed Student t test. dd Number in parentheses is supernatant used as control for calculation of percent migration inhibition. Migration areas of control supernatants 2 to 7 were not significantly different from one another using the 95% confidence limit of the two-tailed Student t test. Differences in percent migration inhibition were always less than 10%.
C. PARVUM ADMINISTRATION AND MIF
VOL. 18, 1977
3, percent migration inhibition of the other control supernatants was always less than 10% (Table 1). Supernatant 3 was therefore chosen for use in the analysis of SLO-specific MIF activity. Migration in chambers containing supernatants of lymphocytes cultured with SLO (experimental group) was compared with migration in chambers containing supernatants of lymphocytes cultured without SLO to which SLO was added after incubation (control group). Analysis of the data with this control supernatant eliminated underlying reactivity to SLO in the guinea pig cells. Three other control supernatants, identical to supernatants 5 to 7 (Table 1) but using SLO as the stimulating antigen, were also compared with supernatant 3. Migration values (10 tests) were not significantly different from one another (see Materials and Methods). MIF activity of canine lymphocytes to SLO antigen. Figure 1 shows the effect of supernatants obtained from canine lymphocytes stimulated by SLO antigen (SLO-S) on the migration of guinea pig peritoneal macrophages compared with the migration in the presence of lymphocyte supernatants from the BCG-vaccinated dogs cultured with PPD. Results from 15 experiments produced a mean migration inhibition, using undiluted supernatants, of 66.8% with a range of 57.4 to 82.1%. A 1:64 dilution inhibited migration by 3%. This resulted in a direct linear relationship between percent migration inhibition with the SLO-rich supernatant and a 10 to 20% decrease in inhibition per twofold dilution. Migration inhibition values for SLO were lower but paralleled the curve obtained with PPD stimulation of lymphocytes from the BCG-vaccinated dog.
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Table 2 demonstrates in a representative experiment that SLO-specific MIF activity inhibited 26.5% macrophage migration at a 1:8 supematant dilution. This is referred to as a titer of 8 MIF units, with 20% macrophage migration as the end point of significance. A 50% inhibition of migration was achieved by a 1:2 dilution of supernatant, and the areas of migration ranged from 3.5 x 103 ILm2 in the presence of undiluted supernatant to 13 x 103 iLm2 at a 1:128 dilution. Enhancement of MIF activity after C. parvum inoculation. Three C. parvum concentrations were administered, each to a group of dogs as a single intramuscular injection. The first dose, 5 mg/m2, elicited no local inflammation or any irritation at the inoculation site. In one of two dogs, the second concentration tested, 50 mg/M2, caused severe swelling and redness, which resulted in drainage. Bacteriological assays revealed no pathogenic organisms at the drainage site. Methylprednisolone was mixed with the 50-mg/M2 suspension of C. parvum for inoculation into a third group, and inflammation at the injection site was totally abrogated. No rise in daily body temperature was detected after any C. parvum inoculations. Clinical parameters, including weight, total and differential cell counts, and liver and blood chemistries, were within normal range. Lymphocytes exposed to SLO from dogs administered the three C. parvum doses (groups 1, 2, and 3) were evaluated for MIF production, twice weekly for 50 days, for comparison with lymphocytes exposed to SLO from untreated dogs (uninoculated control group). All dogs were assayed for MIF production before C. parvum treatment, and the resulting values (10 tests) were pooled with those TABLE 2. Specific inhibition ofguinea pig macrophage migration by supernatants from canine lymphocyte cultures stimulated by SLOa area of L Supernatant dilu- Mean gration (um2 x Migration tion Minhibi-
Superntiont
ti 0.
0
1:2 1:4 1:8 1:16 0
2
4
8
16
32
64
SUPERNATANT OILUTIONS
FIG. 1. MIF activity of supernatants from canine lymphocytes stimulated by SLO (bars) and by PPD stimulation of lymphocytes from a BCG-vaccinated dog (curve). Percent migration inhibition values represent the mean and the range of the mean (vertical lines) of 15 experiments.
1:32 1:64 1:128
3.5 4.7 6.0 7.2 8.0 8.4 9.0 13.0 9.8
64.3 51.5 38.8 26.5 18.4 14.3 8.2 0
Controlb a Representative test; results were similar in 15 experiments. bMacrophages and supernatant from lymphocytes of normal dogs cultured without SLO to which SLO was added after incubation.
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INFECT. IMMUN.
for the uninoculated control group since they were not significantly different from one another using the 95% confidence limit of the two-tailed Student t test. The 1:4 SLO-S was chosen as a representative dilution (see Fig. 2). A dose of 5 mg/m2 (group 1) elicited asignificant (P
INFECTION AND IMMUNITY, Oct. 1977, p. 102-109 Copyright © 1977 American Society for Microbiology
Canine Migration Inhibitory Factor: Effect of Corynebacterium parvum Administration MAYA S. PINEIRO,1 CHARLES A. BOWLES,`* ERNEST C. CUTCHINS,2 AND MALCOLM I. BULLt
Department of Microbiology, Hazleton Laboratories America, Inc., Vienna, Virginia 221801; Department of Biology, Catholic University of America, Washington, D.C. 200642; and Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland 20014
Received for publication 5 April 1977
Peripheral blood lymphocytes from dogs sensitized to streptolysin 0 (SLO) were assayed for migration inhibitory factor (MIF) production by the indirect MIF test, using guinea pig peritoneal exudate cells as the source of macrophages. A specific direct correlation was established between the degree of inhibition of migration and the concentration of SLO-stimulated supernatants from lymphocyte cultures (SLO-S) of untreated normal dogs. Undiluted SLO-S inhibited migration by 66.8%, whereas a dilution of 1:64 elicited a 3% inhibition. In parallel tests, purified protein derivative stimulation of lymphocytes from BCG-vaccinated dogs produced 92.6% inhibition. The effect of Corynebacterium parvum on SLO-specific MIF production was evaluated in three groups of dogs administered a single intramuscular injection of C. parvum at 5 or 50 mg/m2 or 50 mg/mi in suspension with 10 mg of methylprednisolone. Inhibition of migration of macrophages exposed to a 1:4 dilution of SLO-S from dogs inoculated with C. parvum (5 mg/in2) was 33% greater (mean inhibition, 75%) than the same SLOS dilution from uninoculated normal dogs (mean inhibition, 42%) (P < 0.0002). Similarly, lymphocytes from dogs administered 50 mg/m2 caused an enhancement of migration inhibition, with a mean increase of 26% over controls (P < 0.002), whereas a dose of 50 mg/m2 with methylprednisolone produced a 16% increase in migration inhibition (P < 0.05). The administration of C. parvum resulted in a three- to fourfold increase in the SLO-S dilution, which would reduce migration by 20% (MIF titer). This increase peaked between days 20 and 30 and lasted over 50 days post-C. parvum inoculation. These findings indicate that C. parvum specifically increases MIF production by canine lymphocytes in a linear correlation with SLO concentration and suggest its use as a stimulant of canine immunity. Migration inhibitory factor (MIF) is one of a class of lymphokines released by sensitized thymus-derived (T) and bone marrow-derived (B) lymphocytes upon in vitro exposure to specific antigen. These mediators affect the behavior of inflammatory cells, induce mitogenic responses in lymphocytes, participate in cytotoxicity for target cells, promote chemotaxis, aggregate leukocytes, and, in the case of MIF, inhibit the migration of macrophages (6). MIF production is considered an in vitro correlate of cell-mediated immunity (CMI), and the capacity of lymphocytes to produce MIF is used widely as a measure of delayed-type hypersensitivity (11). CMI is important in defense against infection with most viruses and in host resistance to tumors, lymphocytes and macrophages being the effector cells in the recognition and destruction process (12, 19, 29). The role of macrophages in t Present address: 691 Myrtle Lane, Grand Junction, CO 81501.
CMI is intimately linked to and may be directed by sensitized lymphocytes (1). Lymphocyte-released mediators induce a variety of changes in the macrophage periphery and result in increased phagocytic, spreading, and adhesion capacities (28). These changes are believed necessary for the expression of an effective cell-mediated immune response. Experimental tumor systems in rodents have clearly established the role of CMI in host-tumor interactions (27). Nonspecific stimulation of the immune system has been attempted with adjuvants such as BCG and MER (methanol extraction residue of BCG) but has met with limited success due to the variability of strains, number of viable organisms used, and the requirement for repeated regional administration (24-26). Nonspecific immune stimulation with Corynebacterium parvum is now possible with a killed vaccine of standardized potency. C. parvum is a strong immunopotentiator, stimulating antibody 102
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C. PARVUM ADMINISTRATION AND MIF
synthesis and enhancing the immune system's reaction to bacteria and viruses (17). Studies conducted in mice, rats, guinea pigs, and rabbits demonstrate a beneficial influence by C. parvum on the host in a variety of experimental neoplastic systems. Enhanced survival, decreased rate of tumor growth and metastatic dissemination, resistance to chemical carcinogens, and immunity to tumor rechallenge are some of the consequences of C. parvum administration (14, 31, 35). This report describes the effect of intramuscular administration of C. parvum on the CMI response of dogs, using the MIF assay as an indicator of lymphocyte-directed macrophage function. As dogs express naturally acquired sensitivity to streptolysin 0 (SLO), the capacity of lymphocytes to produce MIF after challenge in vitro with SLO antigen was tested to assess the action of C. parvum on their CMI. (This investigation was in partial fulfillment, by M.S.P., for the requirements for the Ph.D. degree in the Department of Biology, Catholic University of America, Washington, D.C.)
103
incomplete Freund adjuvant (Difco Laboratories, Detroit, Mich.). Macrophage migration inhibition assay. A modification of the indirect guinea pig macrophage migration inhibition assay (16) was used. Peritoneal exudate cells were obtained after intraperitoneal inoculation of guinea pigs with 15 ml of emulsified incomplete Freund adjuvant (Difco Laboratories). After 72 h the peritoneal cavity was washed with Waymouth medium containing 2 U of heparin per ml. The collected wash was centrifuged at 100 x g for 10 min, and the pelleted cells were washed in Waymouth medium, again pelleted, and finally suspended at 2 x 107 cells per ml in Waymouth medium. This preparation contained greater than 75% macrophages. Siliconized (Siliclad, Clay-Adams, Parsippany, N.J.) capillary tubes (25 Al; Richard-Allan Medical Industries, Evanston, Ill.) were filled with the cell suspension and flamesealed at one end. The tubes were centrifuged at 200 x g for 10 min and cut at the cell-fluid interface. Duplicate tubes were placed in Mackaness chambers (Berton Plastics, Hackensack, N.J.) and mounted with silicone grease. Triplicate chambers were used for each experimental point, and migration values were averaged. The chambers were assembled by fixing 22-mm, siiconized, round cover glasses on a polycarbonate ring. The supernatants from the lymphocyte cultures MATERIALS AND METHODS were diluted in Waymouth medium and injected into Animals. Adult, male foxhounds housed in indoor the chambers. Each chamber was completely filed, runs and fed standard ration with water available ad the openings were grease-sealed, and the chambers libitum were used throughout the studies. Hartley were incubated at 37°C in 5% CO2 in air for 24 h. strain adult guinea pigs weighing 600 to 800 g (Charles The area of macrophage migration (length by width) River Breeding Laboratories, Wilmington, Mass.) was measured by microscopic reading through a miwere used as a source of peritoneal macrophages. crometer eyepiece (Leitz Limited, Wetzlar, Germany). C. parvum. Dogs were inoculated intramuscularly The percentage of inhibition of migration was calcuin the left hind leg with a single injection of Formol- lated as follows: percent inhibition = 100 - (average killed suspension of C. parvum, batch PX425, 7 mg area of migration in experimental supernatant x (dry weight)/ml (Weilcome Research Laboratory, 100)/(average area of migration in control supernaBeckenham, Kent, England). The dogs were divided tant). Migration inhibition of 20% or greater was coninto three groups (two animals per group), each re- sidered significant (10). Results were evaluated by the ceiving a different concentration of C. parvum. Groups two-tailed Student t test. 1 and 2 were inoculated with 5-mg/ml' (body surface Lymphocyte separation and SLO-specific MIF area) and 50-mg/m2 doses, respectively. Group 3 was production. Lymphocytes were isolated from hepainoculated with 50 mg of C. parvum per m2 mixed rinized canine peripheral blood by centrifugation on with 10 mg of methylprednisolone (Depomedrol, Up- a Ficoll-Hypaque gradient (LSM, Litton Bionetics, john Co., Kalamazoo, Mich.) to avoid the inflamma- Kensington, Md.) by the method of Boyum (7). The tion caused by the high concentration. All doses used lymphocyte layer at the interface was collected and washed twice in Waymouth medium MB 752/1 with were in a total saline inoculum of 1 ml. BCG vaccine. A suspension of 108 viable units of HEPES buffer (N-2-hydroxyethyl piperazine-N'-2Tice BCG vaccine per ml, lot 1L74C (S) 18 (University ethanesulfonic acid; Grand Island Biological Co., of Illinois, Chicago, Ill.), was used to inoculate (0.1 Grand Island, N.Y.). The final cell pellet was susml/site) intradermally five sites in the back of one pended to 107 cells per ml in Waymouth medium with dog. Peripheral blood lymphocytes were incubated for 10ug of gentamicin sulfate per ml (Gentocin, Schering 24 h with 160 ,ug of purified protein derivative (PPD) Veterinary, Kenilworth, N.J.) and 100 yg of disodium per ml (Connaught Laboratories, Toronto, Ont., Can- carbenicillin per ml (Geopen, Roerig, Pfizer Inc., N.Y.). Each preparation contained greater than 90% lymphoada). Skin test. The BCG-vaccinated dog was inoculated cytes. One-milliliter samples of purified lymphocytes intradermally with 250 tuberculin units (5 ,ug in 0.1 at 10' cells per ml were dispensed in 12- by 75-mm ml) of PPD (Mantoux batch CT68, Connaught Labo- culture tubes. Three-tenth milliliter of lyophilized ratories) in the shaved inguinal region of the right SLO reagent (Difco Laboratories; Bacto 0482 stock hind leg. The inoculation site was observed daily for reconstituted to 25 ml with Waymouth medium) was added to each culture tube and incubated for 24 h at duration and redness. Freund adjuvant. The back of one dog was inoc- 37°C in a 5% C02-air atmosphere. Four control superulated intradermally at five sites, at 0.1 ml/site, with natants were examined in 10 standardization experi-
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ments previous to C. parvum treatment. Controls were designed to eliminate underlying reactivity to SLO in the guinea pig cells and dog lymphocytes. Migration of guinea pig exudate cells incubated with SLO alone (I) was compared to the migration area in the presence of medium without the antigen (II). Migration in chambers containing supernatants from normal dog lymphocyte cultures incubated with an equal volume of antigen-free medium (III) and chambers with supernatants from lymphocytes cultured without SLO to which SLO was added after the incubation period (IV) were also compared. As values for migration areas in the four controls were not significantly different from one another using Student's t test, IV was chosen for performing the analysis of percent migration inhibition with regard to SLO and is hereafter referred to as control supernatant. After incubation, supernatants for use in tests of MIF activity were harvested after centrifugation at 200 x g for 10 min. MIF assays were performed before C. parvum injections (10 tests) and twice a week after C. parvum inoculation from day 2 through day 50.
RESULTS
Specificity of macrophage migration. To verify the specificity of the macrophage migration inhibition elicited by a recall antigen in our assay, the known BCG-PPD stimulation system was tested. Lymphocytes from a BCG-inoculated dog were incubated with 160 ,ug of PPD per ml, and the resulting supernatants were evaluated for MIF production by using guinea pig exudate cells. Before lymphocyte procurement, the BCG-vaccinated dog was demonstrated by skin tests to be PPD sensitive. Induration and erythematous skin reactions to PPD were evident at 24 h and were maximal at 72 h after inoculation. The diameter of induration ranged
INFECT. IMMUN.
from 1.5 to 4 cm at the five sites, with a mean of 2.5 cm. Table 1 presents results of MIF assays of undiluted supernatants from PPD-stimulated lymphocytes and the various controls assayed. In five tests the mean migration inhibition produced by lymphocyte supernatants from the BCG-vaccinated dog stimulated with PPD (supernatant 1) was found to be 91.8% with a mean area of migration of 0.9 x 103 ,um2 and a range from 0.6 x 103 to 1.8 x 103 ,m2. This is compared to a mean area of 11.0 x 10 Am2 with a range of 10 x 103 to 15 x 103, m2 for the uninoculated control culture consisting of lymphocytes from an uninoculated dog incubated with an equal dose of PPD (supernatant 2). The specificity of the macrophage migration was further determined by assaying lymphocytes from an uninoculated dog incubated without antigen to which PPD was added after the period of incubation (supernatant 3). When compared to this control, supernatant 1 inhibited migration by 92.6%. Various other control supernatants were evaluated for nonspecific suppression of migration. Macrophages were cultured with: lymphocyte supernatants from a dog inoculated with incomplete Freund adjuvant incubated with PPD (supernatant 4); with PPD in the absence of lymphocyte supernatant (supernatant 5); with lymphocyte supernatants from uninoculated dogs incubated in antigen-free medium (supernatant 6); and macrophages alone (supernatant 7). In all cases (supernatants 2 to 7) migration values were not significantly different from one another using the 95% confidence limit of the two-tailed Student t test. When compared with supernatant
TABLE 1. Guinea pig macrophage migration with undiluted lymphocyte supernatants from control and BCG-vaccinated dogs Supematant source, dog treatMean migration Migration range Migration inhiStimulating antigen (Mg/ml) SiuaigatgnQgm) ment areaa (Am' x 10:') bition (%) (AMm, x 10'.) 1, BCG vaccinated PPD (160) 0.9 ± 0.2" 0.6- 1.8 91.8 (2) 92.6 (3) 2, Uninoculated PPD (160) 11.0 ± 1.3d 10.0-15.0 (2) 9.1 (3) 3, Uninoculated None (PPD added) 12.1 ± 0.6 10.3-14.0 (3) 0 (2) 4, Incomplete Freund inocPPD (160) 12.4 ± 1.1 9.9-16.5 0 (3) ulated 5, None PPD (160) 12.7 ± 0.03 12.0-13.5 0 (3) 6, Uninoculated None 11.2 ± 0.9 9.1-13.5 7.4 (3) 7, None None (medium) 13.0 ± 1.7 9.1-19.1 0 (3) a ± standard error of the mean. bMigration area of supernatant 1 was significantly lower (P < 0.0002) compared to controls 2 to 7, using the two-tailed Student t test. dd Number in parentheses is supernatant used as control for calculation of percent migration inhibition. Migration areas of control supernatants 2 to 7 were not significantly different from one another using the 95% confidence limit of the two-tailed Student t test. Differences in percent migration inhibition were always less than 10%.
C. PARVUM ADMINISTRATION AND MIF
VOL. 18, 1977
3, percent migration inhibition of the other control supernatants was always less than 10% (Table 1). Supernatant 3 was therefore chosen for use in the analysis of SLO-specific MIF activity. Migration in chambers containing supernatants of lymphocytes cultured with SLO (experimental group) was compared with migration in chambers containing supernatants of lymphocytes cultured without SLO to which SLO was added after incubation (control group). Analysis of the data with this control supernatant eliminated underlying reactivity to SLO in the guinea pig cells. Three other control supernatants, identical to supernatants 5 to 7 (Table 1) but using SLO as the stimulating antigen, were also compared with supernatant 3. Migration values (10 tests) were not significantly different from one another (see Materials and Methods). MIF activity of canine lymphocytes to SLO antigen. Figure 1 shows the effect of supernatants obtained from canine lymphocytes stimulated by SLO antigen (SLO-S) on the migration of guinea pig peritoneal macrophages compared with the migration in the presence of lymphocyte supernatants from the BCG-vaccinated dogs cultured with PPD. Results from 15 experiments produced a mean migration inhibition, using undiluted supernatants, of 66.8% with a range of 57.4 to 82.1%. A 1:64 dilution inhibited migration by 3%. This resulted in a direct linear relationship between percent migration inhibition with the SLO-rich supernatant and a 10 to 20% decrease in inhibition per twofold dilution. Migration inhibition values for SLO were lower but paralleled the curve obtained with PPD stimulation of lymphocytes from the BCG-vaccinated dog.
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Table 2 demonstrates in a representative experiment that SLO-specific MIF activity inhibited 26.5% macrophage migration at a 1:8 supematant dilution. This is referred to as a titer of 8 MIF units, with 20% macrophage migration as the end point of significance. A 50% inhibition of migration was achieved by a 1:2 dilution of supernatant, and the areas of migration ranged from 3.5 x 103 ILm2 in the presence of undiluted supernatant to 13 x 103 iLm2 at a 1:128 dilution. Enhancement of MIF activity after C. parvum inoculation. Three C. parvum concentrations were administered, each to a group of dogs as a single intramuscular injection. The first dose, 5 mg/m2, elicited no local inflammation or any irritation at the inoculation site. In one of two dogs, the second concentration tested, 50 mg/M2, caused severe swelling and redness, which resulted in drainage. Bacteriological assays revealed no pathogenic organisms at the drainage site. Methylprednisolone was mixed with the 50-mg/M2 suspension of C. parvum for inoculation into a third group, and inflammation at the injection site was totally abrogated. No rise in daily body temperature was detected after any C. parvum inoculations. Clinical parameters, including weight, total and differential cell counts, and liver and blood chemistries, were within normal range. Lymphocytes exposed to SLO from dogs administered the three C. parvum doses (groups 1, 2, and 3) were evaluated for MIF production, twice weekly for 50 days, for comparison with lymphocytes exposed to SLO from untreated dogs (uninoculated control group). All dogs were assayed for MIF production before C. parvum treatment, and the resulting values (10 tests) were pooled with those TABLE 2. Specific inhibition ofguinea pig macrophage migration by supernatants from canine lymphocyte cultures stimulated by SLOa area of L Supernatant dilu- Mean gration (um2 x Migration tion Minhibi-
Superntiont
ti 0.
0
1:2 1:4 1:8 1:16 0
2
4
8
16
32
64
SUPERNATANT OILUTIONS
FIG. 1. MIF activity of supernatants from canine lymphocytes stimulated by SLO (bars) and by PPD stimulation of lymphocytes from a BCG-vaccinated dog (curve). Percent migration inhibition values represent the mean and the range of the mean (vertical lines) of 15 experiments.
1:32 1:64 1:128
3.5 4.7 6.0 7.2 8.0 8.4 9.0 13.0 9.8
64.3 51.5 38.8 26.5 18.4 14.3 8.2 0
Controlb a Representative test; results were similar in 15 experiments. bMacrophages and supernatant from lymphocytes of normal dogs cultured without SLO to which SLO was added after incubation.
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INFECT. IMMUN.
for the uninoculated control group since they were not significantly different from one another using the 95% confidence limit of the two-tailed Student t test. The 1:4 SLO-S was chosen as a representative dilution (see Fig. 2). A dose of 5 mg/m2 (group 1) elicited asignificant (P
