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-Nondepleted A/J MiceIntratracheally Inoculated T Cell-Depleted or

Infection inLegionella pneumophila

and Reinhard MarreMilorad Susa, Brigita Ticac, Tomislav Rukavina, Miljenko Doric

http://www.jimmunol.org/content/160/1/3161998; 160:316-321; ;J Immunol 

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Legionella pneumophila Infection in IntratracheallyInoculated T Cell-Depleted or -Nondepleted A/J Mice1

Milorad Susa,2* Brigita Ticac,† Tomislav Rukavina,† Miljenko Doric,† and Reinhard Marre*

The inflammatory response and influence of T cell depletion on the pathogenesis of an experimental Legionella infection werestudied. A/J mice were infected with 106 CFU of Legionella pneumophila intratracheally. With this dose all infected animalssurvived the infection and bacteria were cleared from lung, spleen, liver, and kidney within 10 to 11 days, leaving no residualchanges in the affected organs. Inflammatory cells were recruited into the lung on the second day of infection, reaching amaximum on the third day and filling out predominantly the interstitial areas. During the first 3 days after inoculation, mainlymacrophages, B cells, NK cells, and large mononuclear cells of an unknown phenotype were attracted into the lung interstitium,whereas T lymphocytes infiltrated subsequently. During the early phase of infection, serum concentrations of IFN-g, TNF-a,IL-1b, IL-4, and IL-6 but not IL-2 increased dramatically. The cytokine secretion decreased on the third day after infectionalthough bacteria were still present in the lung or even disseminated in different organs. Successful clearance of bacteria fromthe lung was not observed before recruitment of T cells into the lung. In mice depleted of both CD41 and CD81 T cells, controlof infection was impaired and lethality of infection increased. Depletion of either subset left residual antibacterial mechanisms,which, however, were not sufficient to clear the Legionella as rapidly as in undepleted mice. The Journal of Immunology,1998, 160: 316–321.

L egionella pneumophilais an intracellular bacterial patho-gen that causes a serious and often fatal form of pneumo-nia in humans. Alcohol abuse, smoking, old age, or im-

munosuppression increase the susceptibility toL. pneumophilainfection (1–3).

In vivo studies indicate thatL. pneumophilainfections result ina humoral and cell-mediated immune response (4–8). Humoralimmunity probably plays a role as a second line of defense byreducing intrapulmonary growth ofL. pneumophila(9), while cel-lular immunity in concert with cytokines could be essential forresolution of a primary infection (10). IFN-g inhibits intracellularLegionellamultiplication by down-regulation of transferrin recep-tors, thus limiting the amount of available iron (11–13). TNF-acontributes to host defense in experimentalLegionellainfection byfactors that are essentially unknown (14). Although cell-mediatedhost-defense mechanisms seem to be crucial inLegionella infec-tion, kinetics of the cellular response and cytokine release have notbeen studied in detail.

We show thatL. pneumophilainfection is indeed ensued by animmediate production of inflammatory cytokines such as IFN-g,TNF-a, IL-6, and IL-1b, and that the control of infection andclearance ofL. pneumophilafrom the lungs depend on successful

recruitment and unimpaired function of CD4 and CD8 T lympho-cytes in the lung tissue.

Materials and MethodsAnimals

Female pathogen-free A/J mice, 8 to 9 wk of age, were used in all exper-iments. They were housed and cared for in our animal facility according tostandard guidelines.

Bacteria

The L. pneumophilaPhiladelphia strain 1, serogroup 1, (American TypeCulture Collection, Rockville, MD; no. 35133) was cultured for 24 to 36 hon BCYE plates (Merck, Darmstadt, Germany) and harvested with PBS(pH 7.2). The bacteria were washed by centrifugation in sterile saline at4°C and resuspended to give the appropriate concentration.

Inoculation of animals

Mice were inoculated intratracheally according to a previously describedprotocol (15). Briefly, the mice were anesthetized by i.p. injection of ket-amine (2, 5 mg), and the trachea was isolated. A total of 50ml of thebacterial suspension in PBS (106 to 108 L. pneumophila) was inoculateddirectly into the trachea using a 26-gauge needle followed by 10 to 20mlof air. The skin incision was surgically closed. Control animals were in-oculated with PBS only and were sacrificed at different time points.

Quantitation of L. pneumophila in mice

At different time points after inoculation of bacteria, the mice were sacri-ficed by CO2 asphyxia and exsanguinated. The lungs, spleen, liver, andkidneys were subsequently aseptically excised, finely minced, and homog-enized in a tissue homogenizer with 5 ml of sterile distilled water. Thenumber of CFU in the organs was determined by a plate dilution methodusing BCYE agar. After 5 days of incubation at 35°C, 5% CO2, the col-onies were counted and the results were expressed as the number of CFUper organ. Contamination of homogenates was controlled by culturing analiquot of organ homogenate on Mueller-Hinton blood agar for 3 days.

Histopathologic analysis

The pathologic changes and cell recruitment into the lungs of A/J mice inresponse toL. pneumophilawere assessed by light microscopy. At dailyintervals after inoculation, the mice were sacrificed and exsanguinated. Theexcised lungs were inflated and fixed in 10% neutral formalin for 24 h,dehydrated, and embedded in paraffin. Sections (5mm) were cut and

*Department of Medical Microbiology and Hygiene, University of Ulm, Ulm,Germany; and †Department of Microbiology, University of Rijeka, Rijeka,Croatia

Received for publication June 17, 1997. Accepted for publication September18, 1997.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.1 The paper was supported by a grant from the German Research Society (DFG,Ma 864/5.1) and by a grant to Dr. Brigita Ticac from the Federation of EuropeanMicrobiology Societies.2 Address correspondence and reprint requests to Dr. Milorad Susa, Departmentof Medical Microbiology and Hygiene, University of Ulm, Robert Koch Strasse 8,89081 Ulm, Germany. E-mail address: milorad.susa@medizin.uni-ulm.de

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

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stained either with hematoxylin-eosin or with different mAbs using stan-dard immunohistology procedures (anti-monocyte/-macrophage Mac1 Ag,clone M 1/70; anti-granulocyte Ag, clone Gr-1; anti-B lymphocyte AgB220, clone RA3-6B2; anti-CD4 lymphocyte Ag, clone YTS 191.1.2; anti-CD8 lymphocyte Ag, clone YTS 169.4.2; and anti-NK cells, clone DX5).All Abs were obtained from PharMingen (San Diego, CA).

Cytokine assays

The serum cytokine concentrations were determined 2 h after inoculationand on days 1,3,7, and 11 using commercially available mouse ELISAcytokine kits (Endogen, Cambridge, MA).

Modulation of the immune system

To characterize the possible role of distinct T cell populations duringLe-gionella infection, the mice were depleted in some experiments of eitherCD41 lymphocytes, CD81 lymphocytes, or both. For depletion we usedmAbs (anti-CD4, clone 191.1.2 and anti-CD8, clone 169.4.2) purified fromascites using protein G column (HiTrap; Pharmacia, Uppsala, Sweden).The animals were depleted on day 1 before inoculation and once weeklyafter infection with 1 mg of IgG/animal of the respective mAb. The controlgroup of animals received an irrelevant but isotype-identical rat anti-mouseAb (IgG1) at the same time points. The depletion was confirmed by flowcytometry of lymph nodes and spleen cells.

Statistical analysis

Differences in survival times were determined using the Mann-WhitneyUtests, and CFU counts were compared by Student’st test. (16)

ResultsRecruitment of inflammatory cells into the lung induced byinfection with L. pneumophila

Inoculation with 13 106 CFU/animal ofL. pneumophilaled to anevenly distributed cellular infiltration of the infected lung 48 hafter infection. There was no predilection site for the cellular in-filtration. Infiltration was characterized by large, mainly mononu-clear cells, which predominantly filled out the interstitial areas.Three days after inoculation, the interalveolar spaces were heavilystuffed with inflammatory cells so that alveolas virtually collapsed(Fig. 1c). We were not able to observe a marked increase of al-veolar macrophages or infiltration of mononuclear cells into alve-olar spaces at any time point after infection.

On day 7 after inoculation (Fig. 1d) the cellular infiltration re-solved and a normal lung histology developed.

The infiltrating cells were further characterized by immunohis-tology of the lung (Fig. 2). At 24 to 48 h after inoculation ofL.pneumophila, the infiltrating cells were stained by the Mac11 Ab,which recognizes murine monocyte/macrophages and polymor-phonuclear granulocytes. The Mac11 cell infiltration was diffuselydistributed showing no predilection to any anatomical compart-ment of the lung. These cells represent essentially only Mac11

monocyte/macrophages, because a granulocyte-specific Ab (Gr-1)did not stain the cells (data not shown).

Simultaneously with the Mac11 cells, B lymphocytes and NKcells were attracted into lung. B cells segregated nearly exclusivelyaround blood vessels and small bronchiols in the so-called BALT(bronchious associated lymphoid tissue) regions and disappearedat later stages of infection. In contrast, CD41 and CD81 cellscould rarely be detected during the first 2 days postinoculation ofbacteria into the lung, leaving some of the infiltrating cells duringthe early stage of infection undefined.

Systemic production of cytokines during L. pneumophilainfection of A/J mice

Serum levels of IFN-g, TNF-a, IL-1b, IL-2, IL-4, and IL-6 weredetermined, knowing their importance in the resistance of miceagainst intracellular bacteria (17). TheL. pneumophilainfectionresulted in a rapid up-regulation of systemic levels of all the stud-

ied cytokines (Fig. 3) except for IL-2. Pluripotent mediators of theacute phase response such as IL-6 and IL-1b were moderatelyup-regulated as early as 4 to 6 h postinfection, whereas the othercytokines reached maximal serum levels 24 h after infection. Theserum level of cytokines decreased with increasing cell infiltrationof the lung returning to the cytokine level of control sham-infected

FIGURE 1. The histopathology of lung during a L. pneumophila in-fection of A/J mice. Sham-infected lung (a), lung on the first (b), third(c), and (d) seventh days postinoculation of 106 CFU of bacteria. Thesections were stained with hematoxylin and eosin. Note severe inter-stitial infiltration of inflammatory cells. Sham-infected mice did notshow any changes over time. Bar represents 125 mm.

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animals on the third day of infection. An additional wave ofTNF-a and IL-4 was seen 7 days after inoculation of bacteria.

Replication of L. pneumophila in different organs ofintratracheally inoculated A/J mice

After intratracheal inoculation, CFU ofL. pneumophilaincreased 50to 100 times in the lung tissue during the first 2 days of infection and

then slowly decreased again (Table I). This correlated with the kinet-ics of cytokines in the serum. On the third day after inoculation, amoderate dissemination of bacteria to spleen (43 103 CFU), liver(5 3 104), and kidneys (13 104 CFU) was seen.L. pneumophilacould be found in the lungs and sporadically in the liver until day 7after inoculation. At all the examined time points after infection wewere not able to isolateL. pneumophilafrom the blood.

FIGURE 2. Immunohistologic analysis of lung tissue of sham-inoculated mice and of infected mice on days 3 and 7 after intratracheal inoculation of106 CFU of L. pneumophila. Sham-infected mice did not show any changes over time. The cells were stained by DX-5 Abs (NK cells) (a), Mac1(macrophages) (b), B220 (B lymphocytes) (c), CD4 (CD41 T lymphocytes) (d), and CD8 (CD81 T lymphocytes) (e). Bar represents 125 mm.

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The influence of T lymphocyte subpopulations on clearanceof L. pneumophila

The experiments described above showed that the appearance ofCD41 and CD81 T cells correlated with disappearance ofL. pneu-mophila from the lungs. To gain insight into the potential role ofT lymphocytes duringLegionella infection, we made a selectivedepletion of CD41, CD81, or both T cell subsets.

Depletion of CD41, CD81, or CD41 and CD81 T lymphocytesleads to a considerable increase of infection lethality (Fig. 4). Thekinetics of bacterial replication in the lungs of immunocompro-mised mice were similar to that in immunocompetent mice, but thepeak numbers of bacteria were nearly 10 times higher, reaching 1to 2 3 108 CFU/lungs 24 h after infection.

The mice with impaired cellular immunity when inoculated in-tratracheally with 13 106 CFU of L. pneumophilacould survivebut were not able to clear bacteria from the lungs. After havingreached the maximal CFU, bacterial concentrations in the lungsdecreased slowly and persisted for more than 2 wk after infection(Table II).

DiscussionIntratracheal application ofL.pneumophilalead to a pneumoniathat was accompanied by multiplication of bacteria, rapid increaseof cytokines in serum, and recruitment of inflammatory cells intothe pulmonary tissue. The pneumonia resolved spontaneouslywithin 1 wk if the mice were immunocompetent. The infectionmodel used here largely corresponds to that described by Brielandet al. (15). However, in contrast to Brieland et al., who used pul-monary tissue homogenates for immunocytology, we performedimmunohistologic studies to get additional information on the dis-tribution patterns of the different cell types.

It was surprising thatLegionellamore often could be found inthe liver than in the spleen. Perhaps this situation corresponds tothe clinical presentation of Legionnaires’ disease, with a slightelevation of bilirubin and transaminases. One might speculate thatthe Kupffer cells could serve as a suitable host forLegionella.

The course of infection can be divided into an early phase, dur-ing which a rapid bacterial multiplication and inflammatory re-sponse can be observed, and a second phase, beginning on thesecond or third day after infection with a down-regulation of theunspecific inflammatory response and a decrease in the pulmonarybacterial count. The early cellular response was characterized byan interstitial inflammatory reaction consisting mainly of macro-phages, B lymphocytes, and an undefined cell population that wasstained by neither granulocyte-, macrophage/monocyte-, nor lym-phocyte-specific Abs and that might correspond to the mononu-clear/phagocytic cells described by Brieland et al. (15). Additionalstaining with an NK cell-specific Ab revealed that this population,at least in part, was composed of NK cells. NK cells have also beenshown to be the main source of IFN-g in mice experimentallyinfected with Yersinia enterocolitica(18). To the best of ourknowledge, it has not been published before that NK cells repre-sent an early recruited cell population during experimentalLegio-nella infection.

These cells probably represent a first and effective line of de-fense againstL. pneumophilainfection, possibly triggered byLe-gionella LPS and other toxic bacterial constituents. IFN-g, se-creted by T cells and NK cells (17, 18), might directly contributeto intracellular bacteriostasis or killing, either by down-regulationof transferrin receptors (11) or by endogenous nitric oxide (14).Lack of IFN-g induced by disruption of the IFN-g gene impairedclearance ofLegionellaand led to persistent neutrophil recruitmentinto the lungs of BALB/c mice (12). As shown by Brieland et al.

FIGURE 3. Kinetics of TNF-a, IFN-g, IL-1b, IL-4, and IL-6 in re-sponse to L. pneumophila infection. The cytokine concentrations weredetermined using commercially available ELISA kits. The data showmean values and SD obtained from at least six animals.

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(14), TNF-a also helps to control the infection via endogenousnitric oxide. The increase in the number of inflammatory cellsmight be achieved through the effect of TNF-a, which attractsgranulocytes to the site of infection (17). IL-8, mainly produced byendothelial cells, and the monocytic chemoattractant protein 1,produced by pulmonary macrophages and regulated by LPS andinflammatory cytokines (19), also contribute to the increase of in-flammatory cells in the lungs. An increase of systemic IL-2 wasnot detected. This is probably due to the fact that IL-2 is producedby local T cells within the inflammatory region, not affecting theconcentration of this cytokine in the blood stream.

Similar to Brieland et al. (9, 15), we found an increased numberof CD4 and CD8 T cells in the second phase of infection, whencytokines and bacterial counts have already declined. Althoughappearing late in infection, they no doubt significantly contributeto the control of infection during the early phase as well, sincedepletion of CD4 and/or CD8 cells resulted in an increase of in-fection lethality. Furthermore, both T cell subpopulations alsoseem to be relevant for final clearance. This situation is also typicalfor other intracellular pulmonary pathogens, such asChlamydia. Ina Chlamydia trachomatispneumonia mice model (20), IFN-g pro-duction decreased when the mice were CD4 cell depleted, whilethe bacterial burden and lethality increased. The effects of CD8 Tcell depletion were similar but less pronounced. Our experiments,however, are suggestive of a slightly more important role for CD8cells than CD4 cells, since the former are more prominent in theinfected lung tissue and bacterial counts in the lungs tended to belower in CD4- than in CD8-depleted animals.

The two phases of an experimentalLegionella infection alsoreflect the rapid unspecific immune response and the slower de-veloping specific immune response necessary for final eradicationof the infection. CD4 and CD8 T cells play a role in both phasesof infection, as seen from the increased acute lethality during thefirst phase and the long bacterial persistence in the second phase ofinfection in T cell-depleted mice. In the unspecific phase, T cellsmight contribute to host defense by producing IFN-g and IL-6,while in the specific phase they support humoral immunity andspecific T cell-mediated immunity (17). The initial phase mightalso have a parallel to the clinical situation of Pontiac fever, inwhich the flu-like symptoms are characteristic of an overwhelmingproduction of cytokines in response to a massive endotoxinexposure (21).

In this setting of an experimental infection, the immune re-sponse was sufficient to successfully combat the infection. In aclinical situation, however, a specific and unspecific immune re-sponse might be out of balance, thus giving the bacteria a chanceto escape from a pathogen-directed host response. The low endo-toxic activity of LegionellaLPS (22) might insufficiently triggerthe unspecific immune response, and the highly saturated andbranched fatty acids of theL. pneumophilaLPS molecules might

FIGURE 4. Effect of T lymphocyte depletion on survival of A/J micechallenged with different doses of L. pneumophila. The separatedgroups of 10 mice were treated either with an anti-CD4 Ab and anti-CD8 Ab or simultaneously with both mAbs 1 day before intratrachealinoculation of bacteria and once weekly during infection. The controlgroup of mice was treated with an unspecific but isotype-identical ratmAb. The experiments were repeated three times.

Table I. Kinetics of L. pneumophila colony counts in lung, spleen,liver, kidney and blood in A/J mice inoculated intratracheallya

Day AfterInoculation

CFU (3 103) per Organs

Lung Spleen Liver Kidney Blood

0 500 0 0 0 01 9000 0 0 0 03 370 4 55 15 07 60 0 1.6 0 0

11 0 0 0 0 0a Data represent median value of CFU obtained from nine animals assayed at

indicated time points after inoculation of 107 CFU.

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help to resist enzymatic degradation of the bacterial cell wall (23).The metalloprotease cleaves and inactivates cytokines and CD4molecules (25, 26), thus disturbing intercellular communication. Inaddition, the rapid change of metabolic pathways and a group ofso-called early macrophage-induced proteins (26–28) might helpbacteria to multiply undetected or to avert effective host-defensemechanisms.

AcknowledgmentsWe thank Dr. Stipan Jonjic (Department of Histology and Embryology,University of Rijeka, Croatia) for providing mAbs used in our experiments,and to Sonja Weiss for excellent technical assistance.

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losis, Vol. II. S. M. Katz, ed. CRC Press, Boca Raton, FL, p. 159.2. Sheldon, P. A., R. R. Tight, and E. D. Renner. 1985. Fatal Legionnaires’ disease

coincident with initiation of immunosuppressive therapy.Arch. Intern. Med. 145:1138.

3. Cordonnier, C., J. P. Farcet, L. Desforges, C. Brun-Buisson, J. P. Vernant,M. Kuentz, and R. Dournon. 1984. Legionnaires’ disease and hairy-cell leukemia.Arch. Intern. Med. 144:2373.

4. Friedman, A. P., and S. M. Katz. 1981. The prevalence of serum antibodies toLegionella pneumophilain patients with chronic pulmonary disease.Am. Rev.Respir. Dis. 123:238.

5. Neumeister, B., M. Susa, B. Nowak, E. Straube, G. Ruckdeschel, J. Hacker, andR. Marre. 1995. Enzyme immunoassay for detection of antibodies against iso-lated flagella ofLegionella pneumophila. Eur. J. Clin. Microbiol. Infect. Dis.14:764.

6. Friedman, H., R. Widen, and T. Klein. 1983. Cellular and humoral immunity toLegionellaantigen.Clin. Immunol. Newsl. 4:92.

7. Horwitz, M. A. 1983. Cell mediated immunity in Legionnaires’ disease.J. Clin.Invest. 71:1686.

8. Friedman, H., R. Widen, I. Lee, and T. Klein. 1983. Cellular immunity toLe-gionella pneumophilain guinea pigs assessed by direct and indirect migrationinhibition reactions in vitro.Infect. Immun. 41:1132.

9. Brieland, J. K., L. A. Heath, G. B. Huffnagle, D. G. Remick, M. S. McClain,M. C. Hurley, R. K. Kunkel, J. C. Fantone, and C. Engleberg. 1996. Humoralimmunity and regulation of intrapulmonary growth ofLegionella pneumophilainthe immunocompetent host.J. Immunol. 157:5002.

10. Blander, S. J., and M. A. Horwitz. 1991. Vaccination withLegionella pneumo-phila membranes induced cell-mediated and protective immunity in a guinea pigmodel of Legionnaires’ disease.J. Clin. Invest. 87:1054.

11. Nash, T. W., D. M. Libby, and M. A. Horwitz. 1988. IFN-g-activated humanalveolar macrophages inhibit the intracellular multiplication ofLegionella pneu-mophila. J. Immunol. 140:3978.

12. Heath, L., C. Chrisp, G. Huffnagle, M. LeGendre, Y. Osawa, M. Hurley,C. Engleberg, J. Fantone, and J. Brieland. 1996. Effector mechanisms responsible

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13. Gebran, S. J., Y. Yamamoto, C. Newton, T. Klein, and H. Friedman. 1994.Inhibition of Legionella pneumophilagrowth by gamma IFN in permissive A/Jmouse macrophages: role of reactive oxygen species, nitric oxide, tryptophan,and iron (III). Infect. Immun. 62:3197.

14. Brieland, J. K., D. G. Remick, P. T. Freeman, M. C. Hurley, J.C. Fantone, andN. C. Engleberg. 1995. In vivo regulation of replicativeLegionella pneumophilalung infection by endogenous TNFa and nitric oxide.Infect. Immun. 63:3252.

15. Brieland, J., P. Freeman, R. Kunkel, C. Chrisp, M. Hurley, J. Fantone, andC. Engleberg. 1994. ReplicativeLegionella pneumophilalung infection in intra-tracheally inoculated A/J mice.Am. J. Pathol. 145:1537.

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19. Brieland, J. K, M. L. Jones, S. J. Clarke, J. B. Baker, J. S. Warren, andJ. C. Fantone. 1992. Effect of acute inflammatory lung injury on the expressionof monocyte chemoattractant protein-1 (MCP-1) in rat pulmonary alveolar mac-rophages.Am. J. Respir. Cell Mol. Biol. 7:134.

20. Magee, D. M., D. M. Williams, J. G. Smith, C. A. Bleicker, B. G. Grubbs,J. Schachter, and R. G. Rank. 1995. Role of CD8 T cells in primaryChlamydiainfection. Infect. Immun. 63:516.

21. Kaufman, A. F., J. E. McDade, C. M. Patton, J. V. Bennett, P. Skaliy,J. C. Feeley, D. C. Anderson, M. E. Potter, V. F. Newhouse, M. B. Gregg, andP. S. Brachman. 1981. Pontiac fever: isolation of the etiologic agent (Legionellapneumophila) and demonstration of its mode of transmission.Am. J. Epidemiol.114:337.

22. Schramek, S., J. Kazar, and S. Bazovska. 1982. Lipid A inLegionella pneumo-phila. Zentralbl. Bakteriol. Mikrobiol. Hyg. 1 Abt. Orig. A. 252:401.

23. Zahringer, U., Y. A. Knirel, B. Lindner, J. H. Helbig, A. Sonesson, R. Marre, andE. T. Rietschel. 1995. The LPS ofLegionella pneumophilaserogroup 1 (strainPhiladelphia 1): chemical structure and biologic significance.Prog. Clin. Biol.Res. 392:113.

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Table II. Clearance of L. pneumophila from lung after intratracheal inoculation of 107 CFU of bacteria in immunocompetent andimmunocompromised micea

Experimental Group

L. pneumophila Log10 CFU/Lung (log mean 6 SD) day of infection

0 1 3 5 7 11

Control depleted 5.6 6 0.78 6.7 6 0.60 5.0 6 0.58 4.0 6 0.98 0.1 6 0.00 0.0CD4 depleted 5.7 6 0.36 8.2 6 0.17 6.5 6 0.62 6.0 6 0.57 4.2 6 0.52 3.5 6 0.30CD8 depleted 5.6 6 0.88 8.1 6 0.20 6.2 6 0.58 5.1 6 0.51 5.2 6 0.75 4.5 6 0.72CD4/CD8 depleted 5.9 6 0.75 7.6 6 0.21 6.8 6 1.12 6.3 6 0.49 5.3 6 0.51 5.5 6 0.41

a Data show mean value 6 SD of titers from assays repeated at least three times. Five to ten animals per experimental group were analysed.

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