Vol. 57, No. 11

Macrophage-Activating T-Cell Factor(s) Produced in an Early Phaseof Legionella pneumophila Infection in Guinea Pigs

YOSHIHIKO NIKAIDO,"2 SHIN-ICHI YOSHIDA,' YOSHITAKA GOTO,3 YASUO MIZUGUCHI,l*AND AKIO KUROIWA2

Department of Microbiology1 and Second Department of Internal Medicine,2 School of Medicine, University ofOccupational and Environmental Health, Kitakyushu 807, and Department of Cellular Immunology, National

Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141,3 Japan

Received 17 March 1989/Accepted 20 July 1989

Protective immunity of guinea pigs against Legionella pneumophila was studied by infecting the animals witha sublethal dose (about 2 x I0 CFU) of the organism. The bacteria multiplied in the liver, spleen, and lungsup to day 4 after the intraperitoneal infection. The live bacteria in these organs decreased quickly thereafterand were eliminated by day 7. A delayed-type skin reaction and lymphoproliferation of spleen cells toFormalin-killed L. pneumophila were detected from days 5 and 6, respectively, after infection. Peritonealmacrophages obtained from guinea pigs infected 6 days previously inhibited the intracellular growth of L.pneumophila. Antigen-stimulated spleen cell factor prepared from infected guinea pigs inhibited the intracel-lular growth of the organism in macrophages obtained from uninfected animals. Antigen-stimulated spleen cellfactor prepared from spleen cells treated with anti-guinea pig T-cell monoclonal antibody did not inhibitgrowth. The activity of antigen-stimulated spleen cell factor was labile to pH 2 treatment, and the factor couldnot be absorbed by L. pneumophila antigen, suggesting that it contains gamma interferon. Our data show thatT-cell-mediated immunity begins to work from an early period of infection with L. pneumophila in guinea pigs.

Legionella pneumophila is a facultative intracellular bac-terium that multiplies in macrophages of both humans (21,34) and rodents (27, 44). The organism is resistant tolysozyme (1), inhibits phagosome-lysosome fusion of humanmonocytes (19), inhibits acidification of phagosome (20), andproduces a cytotoxin(s) (14, 15) and proteinases (32, 40).These characteristics of L. pneumophila are thought to bepathogenic factors which enable it to proliferate in macro-phages and cause pneumonia. Although Legionella infectionwas initially reported in humans (12), guinea pigs presentmorbid symptoms and pathological changes in organs similarto those presented by humans after infection. Therefore,they are widely used as an animal model of human legionel-losis (3, 8, 9, 26).

Cell-mediated immunity is thought to play the most im-portant role in protecting against L. pneumophila. Horwitz(18) reported that cell-mediated immunity is established inhumans after infection with L. pneumophila. Human alveo-lar macrophages activated by gamma interferon (IFN-y) caninhibit intracellular multiplication of the organism (35). Fur-thermore, glycoprotein extract of Klebsiella pneumoniae(37) and concanavalin A-stimulated human mononuclear cellsupernatant (22) activate human monocytes, and L. pneu-mophila cannot multiply in the activated monocytes. Inguinea pigs, establishment of a delayed-type skin reactionagainst Formalin-killed L. pneumophila (4, 28) and macro-phage activation to inhibit intracellular growth (16, 45) havealso been reported. However, the nature of the delayed-typeskin reaction and its relation to macrophage activation havenot been well analyzed (42).When guinea pigs were infected with a sublethal dose of L.

pneumophila, elimination of the bacteria from organs beganday 5 after the intraperitoneal (i.p.) infection. This phenom-enon, observed in the early phase of infection, led us tostudy the nature of cell-mediated immunity to Legionella

* Corresponding author.

infection, especially regarding spleen cell factors producedduring this stage.

MATERIALS AND METHODSBacteria. L. pneumophila Philadelphia-1 (ATCC 33152)

was donated by the Centers for Disease Control, Atlanta,Ga., in 1980. The organism was passaged once in guinea pigsbefore it was used in this study; i.e., it was inoculated i.p.into Hartley strain guinea pigs. Fresh isolates were obtainedfrom the spleen on day 7 postinoculation and were grownonce on charcoal-yeast extract (CYE) agar plates (11) andstored at -80°C in tryptic soy broth (Difco Laboratories,Detroit, Mich.) supplemented with 20% (vol/vol) glycerol.Formalin-killed L. pneumophila was prepared as describedby Friedman et al. (13). Briefly, the organism was culturedon CYE agar plates for 4 days and harvested with saline.After being washed, the bacterial cells were treated with0.5% Formalin in saline at 37°C for 24 h. We confirmed thatall bacteria were killed by the treatment. Killed bacteriawere stored at -80°C until use.

Animals. Female outbred Hartley strain guinea pigs were

purchased from Seiwa Experimental Animal Institute, Oh-ita, Japan. Guinea pigs weighing 400 to 500 g were used forexperiments.

Infection. Guinea pigs were injected i.p. with a sublethaldose (2 x 104 CFU) of L. pneumophila (50% lethal dose, 7.6x 104 CFU [44]). They exhibited fever (at least 1°C eleva-tion) and weight loss (about 10%) by day 4 after infection,but they recovered completely by day 7.Growth of L. pneumophila in organs. A total of 21 guinea

pigs were infected sublethally and were sacrificed in groupsof three by cardiac puncture at 3 h and at 1, 2, 3, 4, 5, and 7days after infection. The number of organisms in the rightlobe of the liver, spleen, and both lungs were counted onCYE agar plates after appropriate dilution of the homoge-nized suspension.

Skin tests. Skin tests were carried out 3, 5, 7, 14, and 21

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days after sublethal infection. Formalin-killed L. pneumo-phila was suspended in saline at a concentration of 109bacterial particles per ml, and 0.1 ml of the suspension wasinjected intradermally into the shaved dorsal skin. Erythemaand induration at the reaction sites were measured 24 and 48h after the elicitation.Antibody titration. Antibodies in sera against L. pneumo-

phila were measured by the indirect immunofluorescencetest (41).

Lymphoproliferative assay. A lymphocyte proliferationassay was performed by the method described by Breimanand Horwitz (4) after slight modification. Spleens wereobtained from guinea pigs which had been infected suble-thally 4, 6, 8, 14, and 21 days before the experiment. Single-cell suspensions of splenocytes were prepared by passingthem through metal mesh. Splenic lymphocytes were sus-pended in RPMI 1640 medium (Nissui Pharmaceutical Co.Ltd., Tokyo, Japan) containing 100 U of penicillin per ml,100 ,ug of streptomycin per ml, and 5% (vol/vol) heat-inactivated fetal calf serum (FCS; GIBCO Laboratories,Grand Island, N.Y.), and suspensions were adjusted to aconcentration of 108 cells per ml. Cells were incubated with108 bacterial particles of Formalin-killed L. pneumophila asan antigen in a total volume of 100 ,ul in a 96-well, flat-bottomtissue culture plate (Falcon 3072; Becton Dickinson Lab-ware, Lincoln Park, N.J.). As a control, lymphocytes wereincubated without the antigen or with concanavalin A (typeIV; Sigma Chemical Co., St. Louis, Mo.) at a concentrationof 5 ,ug/ml. These cells were incubated for 24 h at 37°C in 5%CO2 (CO2 incubator). In the last 2 h of incubation, 0.25 ,uCiof [3H]thymidine (Amersham Japan, Ltd., Tokyo) wasadded to each well. Cells were harvested with a multisampleautomatic cell harvester (Labo Science Co., Ltd., Tokyo,Japan), and filter mats with cells were placed in scintillationvials containing 6 ml of EX-H scintillation cocktail (WakoPure Chemical Industries, Ltd., Osaka, Japan). Beta-particleemission was measured with a beta scintillation counter(Beckman Instruments, Inc., Fullerton, Calif.). The stimu-lation index (SI) was calculated by the following formula: SI= cpm in the presence of antigen/cpm in the absence ofantigen.

Anti-T-cell monoclonal antibody. Monoclonal antibodyagainst guinea pig T lymphocytes (8BE6) was kindly donatedby Joe Chiba, National Institute of Health, Tokyo, Japan (6).This monoclonal antibody specifically lyses guinea pig pe-ripheral T cells in the presence of rabbit complement.Splenic lymphocytes (108 cells) were suspended in 5 ml ofRPMI 1640 medium containing 5% (vol/vol) heat-inactivatedFCS, and 10 RI of 8BE6 plus 0.5 ml of rabbit complement(Cedarlane Laboratories Ltd., Hornby, Ontario, Canada)were added. After incubation at 37°C for 60 min withoccasional shaking, the spleen cells were washed three timeswith Hanks balanced salt solution (Nissui) and suspended in10 ml of RPMI 1640 containing 5% (vol/vol) heat-inactivatedFCS. The number of viable cells was counted by the trypanblue dye exclusion method, using a hemacytometer. About30% of the splenic lymphocytes were lysed by the treatment.

Preparation of ASSF. Splenic lymphocytes were preparedfrom normal guinea pigs or guinea pigs infected sublethally4, 6, and 22 days before the experiments. Spleen cells(107/ml) were cultured with Formalin-killed L. pneumophila(108/ml) in RPMI 1640 medium, using tissue culture flasks(no. 25100; Coming Glassworks, Corning, N.Y.), for 48 h at37°C in a CO2 incubator. After incubation, the culturesupernatants were collected by centrifugation at 500 x g for15 min. After the supernatants were passed through a

0.45-,um-syringe filter (Corning), they were concentrated bypassage through Diaflo ultrafiltration membranes (cuttingmolecular weight, 5,000; Amicon Corp., Danvers, Mass.) toa final volume of 1/10. After concentration, the supernatantswere dialyzed twice in phosphate-buffered saline for 12 h andonce in RPMI 1640 medium for 12 h. The dialyzed factorswere passed through a 0.45-,um-syringe filter for sterilizationand stored at -80°C until use. Medium control of antigen-stimulated spleen cell factor (ASSF) was prepared by con-centrating and dialyzing in RPMI 1640 containing 5% (voVvol) heat-inactivated FCS as mentioned above.

Acid treatment of ASSF. Acid treatment of ASSF wascarried out by the method of Zlotnik et al. (46). Briefly, 30 mlof ASSF was dialyzed for 24 h against 1,000 ml of glycine-hydrochloride buffer (0.1 M, pH 2.0; containing 0.15 MNaCl), followed by dialysis for 48 h against phosphate-buffered saline and a final dialysis against RPMI 1640 for 12h. The factor was sterilized by passing it through a 0.45-pum-syringe filter.Antigen absorption of ASSF. ASSF was treated with For-

malin-killed L. pneumophila antigen. About 0.5 g of Forma-lin-killed L. pneumophila was mixed with 3 ml of ASSF andincubated at 37°C for 30 min. After incubation, the ASSFswere collected by centrifugation at 500 x g for 15 min andpassed through a 0.45-,um-syringe filter to sterilize and toremove antigen particles.

Bactericidal activity of ASSF. Direct bactericidal action ofASSF was tested by the following procedures. A freshculture of L. pneumophila was washed once in sterilephosphate-buffered saline and suspended in RPMI 1640medium at a concentration of ca. 107 CFU/ml. The bacterialsuspension (0.1 ml) was mixed with 0.9 ml of ASSF in a testtube, and the mixture was incubated at 37°C for 30, 60, and120 min. After incubation, the mixture was diluted appropri-ately and inoculated on CYE agar plates to determine thenumber of viable cells. As a control, RPMI 1640 mediumcontaining 50% (vol/vol) heat-inactivated FCS was used.

Intracellular growth of L. pneumophila in peritoneal mac-rophages. Intracellular growth of L. pneumophila in perito-neal macrophages from infected guinea pigs and proteosepeptone-induced macrophages incubated with ASSFs wereexamined after in vitro phagocytosis. This assay was carriedout by the method described by Yoshida et al. (43, 45) aftersome modifications.To estimate macrophage activation by i.p. infection with

L. pneumophila, macrophages were collected from guineapigs infected with 2 x 104 CFU of live L. pneumophila 6, 8,15, and 22 days before examination. Collected peritonealexudate cells were washed, and then the number of viablecells was determined in a hemacytometer by the trypan bluedye exclusion method. Differential cell counts were done onWright- and Giemsa-stained smears of peritoneal exudatecells. After adjustment of the macrophage concentration to 2x 106 cells per ml, 0.5 ml of the cell suspension was placedin each well of a 24-well, flat-bottom tissue culture plate(Falcon 3047; Becton Dickinson Labware) and the plate wasincubated for 90 min at 37°C in a humid atmosphere of 5%CO2 in room air. Nonadherent cells were washed out withphosphate-buffered saline.

In vitro phagocytosis was carried out by adding 0.5 ml ofthe bacterial suspension (2 x 106 CFU/ml) to each well andincubating at 37°C in a humid atmosphere of 5% CO2 for 90min. After washing, 0.5 ml of RPMI 1640 containing 5%(vol/vol) heat-inactivated FCS was added to each well. After0, 24, and 48 h of incubation, 0.5 ml of sterile distilled waterwas added to wells and adherent cells were scraped with a

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TABLE 1. Delayed-type hypersensitivity against Formalin-killedL. pneumophila in sublethally infected guinea pigs

Days after Time (h) Extent (mm + SD; n = 9) of:Dasnfetern afterinfection elicitation Erythema Induration

Control 24 NDa ND48 ND ND

3 24 0.6 ± 1.3 ND48 ND ND

5 24 9.5 ± 0.6 4.0 ± 0.848 4.5 ± 1.0 ND

7 24 12.9 ± 3.1 7.1 ± 1.448 8.4 ± 1.8 5.6 ± 2.2

14 24 9.3 ± 1.5 4.5 ± 1.348 8.8 ± 0.9 8.5 ± 0.6

21 24 10.0 ± 1.6 8.8 ± 0.948 9.3 ± 0.9 9.5 ± 0.6

a ND, Not detected.DAYS AFTER INFECTION

FIG. 1. Growth of L. pneumophila in the liver, spleen, and lungsof sublethally infected guinea pigs. The CFU of L. pneumophila inliver (A), spleen (A), and both lungs (0) were counted at intervalsafter guinea pigs were infected i.p. with 2 x 104 CFU of theorganism. This is representative of five experiments.

rubber policeman. After sonication, the cell suspension wasdiluted appropriately, and 0.1 ml was inoculated onto CYEagar to determine the number of L. pneumophila.To examine the effect of ASSF on the intracellular growth

of the organism, peritoneal exudate cells were collected bylavaging the peritoneal cavity of healthy animals which hadbeen injected with 10 ml of 10% (vol/vol) proteose peptoneno. 3 (Difco) as irritant. Preparation of macrophage mono-layers and in vitro phagocytosis were carried out as men-tioned above. After phagocytosis, 0.5 ml of ASSF was addedto each well. After 0, 6, 12, 18, 24, and 48 h of incubation, thenumber of intracellular bacteria were counted on CYE agarplates.

Statistics. Data were analyzed by analysis of variance andStudent's t test. A P value of <0.05 was significant.

RESULTS

Growth of L. pneumophila in the liver, spleen, and lungs.Guinea pigs were infected i.p. with a sublethal dose (2 x 104CFU) of L. pneumophila Philadelphila-1, and CFU in theorgans were counted at intervals. Figure 1 is representativeof five experiments which showed similar growth and elim-ination patterns. At 3 h after infection, the liver and spleencontained about 2 x 103 CFU of the organism. CFU in theseorgans reached a maximum 4 days after the infection: about8 x 105 in the liver and spleen and about 105 in the lungs.Bacterial numbers decreased immediately thereafter, andthe organism became undetectable (<102 CFU) 7 days afterinfection.

Delayed skin reaction against Formalin-killed L. pneumo-phila. To study cutaneous delayed-type hypersensitivity, weinjected 0.1 ml of Formalin-killed bacterial suspension intothe dorsal skin of infected animals. Erythema and indurationwere measured at 24 and 48 h after the elicitation. Erythema

began to appear after 5 days of infection (Table 1). However,at 48 h it was significantly smaller than at 24 h in guinea pigsduring the early stage of a positive skin test (5 and 7 daysafter infection). Induration of 48 h at the skin reaction siteswas also smaller than the erythema at day 5 or 7 of infection,but reached almost the same size as erythema after day 14 ofinfection.

Proliferative response of spleen cells against Formalin-killedL. pneumophila. To assess the time course of the prolifera-tive response of lymphocytes in infected guinea pigs, spleencells taken at various stages of infection were cultured invitro with Formalin-killed L. pneumophila, and proliferationwas assayed by [3H]thymidine uptake. The SI of spleen cellssignificantly increased beginning day 6 after the sublethalinfection, and the positive proliferative response continueduntil day 22 after infection (Table 2).

Spleen cells of guinea pigs infected 6 days before theexperiment were treated with anti-T-cell monoclonal anti-body. Treated spleen cells did not show any proliferativeresponse after stimulation with Formalin-killed L. pneumo-phila or concanavalin A (Table 3).Humoral immune response to L. pneumophila in sublethaHly

infected guinea pigs. Production of serum immunoglobulin Gwas assayed by the indirect immunofluorescence method,using fluorescein isothiocyanate-labeled goat anti-guinea pigimmunoglobulin G. Antibodies against L. pneumophila weredetected from day 6 after infection and reached a maximum10 days after infection (titer, 8 to 64) (Fig. 2).

Intracellular growth of L. pneumophila in peritoneal mac-rophages obtained from infected guinea pigs. The ability ofmacrophages to inhibit intracellular growth of L. pneumo-phila was measured in vitro. Macrophages were lavagedfrom the peritoneal cavities of normal guinea pigs or guineapigs infected sublethally 6, 8, 15, and 22 days before theexperiment. After in vitro phagocytosis, intracellular growthof L. pneumophila in these macrophages was measured at 24and 48 h of incubation (Fig. 3). The intracellular bacterialgrowth was significantly inhibited in the macrophages ob-tained from guinea pigs infected 4 and 8 days before theexperiment, after 24 (P < 0.01) and 48 (P < 0.001) h of invitro infection. The organism multiplied 10-fold by 24 h inperitoneal macrophages from both normal guinea pigs and

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TABLE 2. Proliferative response of spleen cells against Formalin-killed L. pneumophila

Days after Animal None Concanavalin A, 5 jig/ml L. pneumophila Meaninfection no. (cpm ± SD) cpm + SD Si cpm ± SD Si SI + SD

Control 1 423 ± 29 1,556 ± 45 2.9 1,710 ± 279 4.0 5.0 ± 1.02 532 ± 190 1,258 ± 155 3.2 2,271 ± 249 5.23 347 ± 109 3,153 ± 176 9.0 2,045 ± 217 5.9

4 1 131 26 465 123 3.6 231 29 1.8 3.3 ± 1.82 197 ± 62 271 ± 127 1.4 561 ± 247 2.93 223 ± 5 739 ± 206 3.3 1,169 ± 101 5.3

6 1 249 ± 73 459 ± 48 1.8 5,324 + 652 21.4 27.8 ± 6.6a2 963 ± 84 2,639 ± 174 2.7 26,396 ± 2,978 27.43 271 ± 92 1,452 ± 278 5.4 9,357 ± 1,249 34.6

8 1 778 ± 143 5,524 ± 1,038 7.1 9,695 ± 708 12.5 16.7 3.7a2 325 ± 37 3,191 ± 104 9.8 5,800 ± 1,467 17.93 354 ± 71 4,586 ± 302 12.9 6,984 ± 1,746 19.7

15 1 814 ± 65 8,418 ± 1,054 10.3 15,009 ± 959 18.4 22.9 5.Oa2 1,037 ± 167 4,929 ± 735 4.8 22,811 ± 972 22.03 677 ± 8 12,469 ± 1,778 18.4 19,147 ± 1,815 28.2

22 1 932 ± 160 3,373 ± 1,236 11.0 13,013 ± 1,132 14.0 20.1 ± .02 469 ± 67 5,633 ± 1,026 12.0 8,765 ± 2,596 18.73 291 ± 79 5,687 ± 822 19.5 8,071 ± 400 27.7

a p < 0.01 compared with controls.b p < 0.05 compared with controls.

guinea pigs infected 15 and 22 days prior to the experiment. ASSF. We studied the origin of the macrophage-activatingAt 48 h, CFU of the organism in peritoneal macrophages factor (MAF) in ASSF. Spleen cells obtained from guineafrom normal guinea pigs multiplied 100-fold, but multiplica- pigs infected 6 days previous to the examination weretion in peritoneal macrophages from guinea pigs infected 15 treated with anti-guinea pig T-cell monoclonal antibody andand 22 days before the experiment was less, although the rabbit complement. Then ASSF was prepared from thedifference was not significant. spleen cells and its activity was tested in the same way as

Effects of ASSF on intracellular growth of L. pneumophila. explained above. ASSF prepared from T-cell-depletedSpleen cells were obtained from guinea pigs infected 4, 6, or spleen cells did not inhibit the intracellular growth of L.22 days before the experiment, and ASSF was prepared by pneumophila (P < 0.01) (Fig. 5).stimulation in vitro with Formalin-killed L. pneumophila. Further characterization of ASSF. To test whether ASSFProteose peptone-induced peritoneal macrophages were pre- contains IFN--y or antibody, ASSF prepared from guineapared from normal guinea pigs; after in vitro phagocytosis, pigs infected 6 days before the examination was subjected toASSF was added to the macrophage culture. Intracellular acid treatment or antigen absorption. The growth-inhibitinggrowth of L. pneumophila was measured after 6, 12, 18, 24, ability of ASSF was significantly (P < 0.01) abrogated byand 48 h of incubation (Fig. 4). ASSF obtained from guinea treatment at pH 2 (Fig. 5). Compared with the mediumpigs infected 4, 6, or 22 days prior to the experiment was control, however, pH 2-treated ASSF still possessed theeffective in causing macrophages to restrict significantly (P activity to inhibit intracellular multiplication of the organism< 0.01) the intracellular growth of L. pneumophila. In significantly (P < 0.05). Absorption of ASSF with Formalin-comparison with a medium control, ASSF prepared from killed L. pneumophila did not change the macrophage-normal guinea pigs also restricted multiplication of L. pneu- activating ability of ASSF. An indirect immunofluorescencemophila to some degree. assay showed that no ASSF preparation contained anti-L.

Effects of anti-guinea pig T-cell monoclonal antibody treat- pneumophila antibodies. Furthermore, no bactericidal activ-ment of spleen cells on macrophage-activating activity of ity was detected in ASSF preparations (data not shown).

TABLE 3. Proliferative response of T-cell-depleted spleen cells

Expt T-cell None Concanavalin A, 5 jig/ml L. pneumophilaelimination (cpm ± SD) cpm ± SD SI cpm ± SD SI

1 - 1,208 ± 50 20,490 ± 3,449 17.0 10,704 ± 1,730 8.8+ 1,789 ± 166 5,245 ± 48 2.9 4,819 ± 187 2.7

2 - 2,066 ± 394 12,388 ± 2,621 6.0 18,410 ± 2,431 8.9+ 3,098 ± 190 3,785 ± 344 1.2 5,198 ± 493 1.6

3 - 1,325 ± 128 3,271 ± 7 2.5 15,899 ± 2,206 12.0+ 1,943 ± 466 1,183 ± 201 0.6 2,589 ± 126 1.3

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FIG. 2. Humoral immune response to L. pneumophila in suble-thally infected guinea pigs. Production of serum immunoglobulin Gwas assayed by the indirect immunofluorescence method, usingfluorescein isothiocyanate-labeled goat anti-guinea pig immunoglob-ulin G.

DISCUSSION

When guinea pigs were infected i.p. with a sublethal doseof L. pneumophila, the bacterial number in the liver, spleen,and lungs increased up to day 4 after infection. However, thebacterium was eliminated from these organs by day 7 afterinfection (Fig. 1). To understand clearly the protectivemechanisms working in such an early phase of infection, weinvestigated skin reaction, splenic lymphocyte proliferation,and ability of peritoneal macrophages to inhibit intracellularbacterial growth. Erythema and induration were detected asearly as 5 days after infection (Table 1). A splenic lympho-cyte proliferation test showed that the SI significantly in-creased from day 6 (Table 2) and T-cell-depleted spleen cellsdid not proliferate after antigen stimulation (Table 3). Themacrophages obtained from the animals 4 days after infec-tion inhibited the multiplication of intracellular bacteria (Fig.3), indicating that macrophages are already activated by day4. These results imply that the cell-mediated immune re-sponse is playing an important role in the protection of L.pneumophila at an early phase of infection. To analyze thisphenomenon further, ASSF was obtained from infectedguinea pigs. The results show that ASSF taken during theearly phase of infection had MAF which inhibited intracel-lular multiplication of the bacterium. The MAF was shownto be produced by T cells (Fig. 5). These results indicate thatT-cell-mediated immunity plays an important role in hostdefense against Legionnaires disease.

During the past 20 years, a number ofMAF of T-cell originhave been reported. The activated macrophages are effectivein killing or inhibiting intracellular growth of some intracel-lular bacteria (29, 30), protozoa (36), and tumors (10). Since1980, it was made clear that IFN-y produced by T lympho-cytes is a major component ofMAF (33, 36, 38). ConcerningL. pneumophila, MAF activity in humans was reported byHorwitz (18), and his colleagues also reported that humanrecombinant IFN--y activates human alveolar macrophages

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FIG. 3. Intracellular growth of L. pneumophila in peritonealmacrophages obtained from infected guinea pigs. Macrophages werelavaged from the peritoneal cavity and cultured in vitro. After invitro phagocytosis, intracellular growth of L. pneumophila wasmeasured at 24 and 48 h of incubation. Symbols: macrophagesobtained from guinea pigs infected 4 (A), 8 (0), 15 (U), and 22 (A)days before the examination; (0) normal macrophages.

to inhibit intracellular growth of L. pneumophila (35). In ourguinea pig system, MAF in ASSF was labile to low-pHtreatment, was not absorbed by Legionella antigen (Fig. 5),and did not have direct bactericidal activity. This evidenceindicates that the main component of MAF activity is alsoIFN--y. But MAF activity could not be abrogated completelyby pH 2.0 treatment (Fig. 5), possibly because our ASSFmight also have included factors other than IFN--y or it mightstill have contained residual IFN--y even after pH 2.0 treat-ment. Recently, it has been reported that IFN--y alonecannot activate macrophage bactericidal activity againstListeria monocytogenes (5) and some mycobacteria (7, 39).A tremendously augmented SI was detected on day 6 of

immunization. It is reported that L. pneumophila itself hasan adjuvant activity (31). This adjuvant activity will supportboth the high SI in an early phase of the infection and anearly establishment of delayed hypersensitivity and macro-phage activation.Although some investigators (4, 13, 28) have reported that

a delayed skin reaction is detected in Legionella infection,its nature is not well characterized. The important observa-tion we made is that a delayed skin reaction is detected fromas early as 5 days after infection and its time coursecorresponds to the onset of bacterial elimination and mac-rophage activation. Delayed skin reaction elicited on day 5revealed erythema accompanied by negligible induration(Table 1). The erythema was also elicited by Formalin-killedL. pneumophila Bloomington (serogroup 3) and Los Angeles(serogroup 4) 5 days after immunization with L. pneumo-phila Philadelphia-1 (serogroup 1). But significant erythemawas not elicited by Formalin-killed Escherichia coli, Listeriamonocytogenes EGD, or Salmonella typhimurium LT-2(unpublished data). Also, the macrophage migration inhibi-

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FIG. 4. Effects of ASSF on intracellular growth of L. pneumo-phila. ASSF was prepared by stimulation in vitro with Formalin-killed L. pneumophila. Proteose peptone-induced peritoneal macro-phages were prepared, and after in vitro phagocytosis, ASSF wasadded to the macrophage culture. The intracellular growth of L.pneumophila was measured after 6, 12, 18, 24, and 48 h of incuba-tion. Symbols: ASSF from guinea pigs infected 4 (A\), 6 (0), or 22([1) days before examination; (G) ASSF from normal guinea pigs;(0) medium control.

tion test was negative in the early phase of infection (unpub-lished observation). From these characteristics, we thinkJones-Mote-type hypersensitivity is established in the earlyphase of infection (17).ASSF from normal guinea pigs, which was stimulated by

Formalin-killed L. pneumophila during incubation, inhibitedintracellular multiplication to some degree. It is possible thatnormal spleen cells produce MAF by the in vitro antigenstimulation.The macrophages obtained from guinea pigs 22 days after

infection could not inhibit intracellular multiplication, butASSF from guinea pigs 22 days after infection could activateproteose peptone-induced macrophages to inhibit intracellu-lar multiplication (Fig. 4). This discrepancy might be be-cause the duration of macrophage activation in vivo ininfected guinea pigs was not very long, acquired T-cellmemory did not disappear during that period, and MAF wasproduced in vitro during this stage.

Antibodies in sera were detected from day 6 after infec-tion. Horwitz and Silverstein reported that antibody andcomplement promote a modest amount of phagocytosis ofvirulent L. pneumophila by human polymorphonuclear leu-kocytes and monocytes, but these humoral componentscannot cause phagocytes to inhibit intracellular multiplica-tion of the organism (23, 24). In the absence of cell-mediatedimmunity, antibody may not serve the cause of host defensevery effectively, because antibody may promote uptake of L.pneumophila into nonactivated monocytes in which thebacteria can multiply. In the presence of cell-mediatedimmunity, antibody may serve host defense by promotinguptake of L. pneumophila into activated macrophages,which inhibit the intracellular multiplication of the bacte-

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FIG. 5. Effect of T-cell depletion, acid treatment, and antigenabsorption on activity of ASSF. Symbols: (0) medium control; (A)ASSF from T-cell-depleted splenic lymphocytes; (x) ASSF treatedat low pH; (U) ASSF from spleen cells of uninfected guinea pigs; (l)ASSF treated with a large amount of legionella antigen; (0) ASSFfrom guinea pigs infected 6 days prior to examination.

rium. Such activated macrophages may assist the host inclearing L. pneumophila from infected tissues.

Dissociation between delayed-type hypersensitivity andresistance to Listeria monocytogenes (2) or Mycobacteriumlepraemurium (25) is reported in mice. Soon we intend tostudy whether guinea pig delayed-type hypersensitivity andprotective immunity against L. pneumophila are mediatedby the same T-cell clone and the same lymphokine.

ACKNOWLEDGMENTS

We are grateful to Joe Chiba for kindly donating anti-guinea pigT-cell monoclonal antibody (8BE6). We thank Midori Ogawa andKumiko Mizue for technical assistance.

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