Folia Microbiol. 41 (4), 309-314 (1996)

Porcine Interferon- , Inhibits the Growth of Legionella pneumophila in WiREF Cells in E. EBERL-GREGORIO a, B. FILIPI(~ a*, S. ROZMAN b, A. CENCI~ ~c, and B. DRINOVEC a

alnstitute for Microbiology and Immunology, Medical Faculty, 1105 Ljubljana, Slovenia bBIA Ltd., 1000 Ljubljana, Slovenia CFaculty of Agriculture, 2000 Maribor, Slovenia

Vitro

Received January 15, 1996 Revised version May 28~ 1996

ABSTRACT. The intraceUular growth of Legionella pneurnophila in WiREF Ohristar rat embryonal fibroblast) cells was inhib- ited by porcine interferon-~r The effect was compared with that of different human interferons (~t and 7). The growth inhibition was dose-dependent and required the pretreatment of WiREF cells with interferon. The development of an antibacterial state of the cells was observed. When interferon was added together with bacteria or 1 d after the infection there was no inhibition. Also, there was no direct antibacterial effect of the interferon. In addition, cell pretreatment with a combination of interferon and antibiotics failed to show a synergistic effect.

Bacteria in the genus Legionella are Gram-negative aerobic bacilli which are pathogenic for man. The first described outbreak of pneumonia in Philadelphia in 1976 (Fraser et al. 1977) led to the recognition of the clinical entity of Leglonaire's disease together with the identification of Legionella pneumophi la as an etiological agent. The legionellae are unusual in the sense that they will not grow in routine bacteriological media, but require a special one (BCYE-Alpha) (Paculle et aL 1980). They will grow and multiply in vivo and in vitro in phagocytes (Jacobs et al. 1984; Payne and Horwitz 1987; Yamamoto et al. 1994) and also in vitro in other cells that are not professional phagocytes (Rinaldo et al. 1981; Daisy et al. 1981). In assessing the virulence of a test strain of Legionella pneumophila, it is convenient to use the guinea pig assay system or fertile chicken eggs to determine the 50 % lethal dose. However, these systems do not easily lend themselves to routine screening. Because of this, an in vitro virulence assay on mouse cells L929 was developed (Fernandez et al. 1989).

Interferon (IFN) can inhibit the growth of different intracytobionts in cell cultures and in in vivo conditions. These include chlamydiae and rickettsiae as well as protozoa (Gober et al. 1972; Jahiel et al. 1968). The antibacterial effect is not restricted to just one type of IFN. In the human system, the experiments were focused on monocytes/macrophages, and much fewer on different cell lines. Pre- treatment of these cells with HulFN u or ), leads to growth and multiplication restriction of L. pneumo- phila (Nash et al. 1988). It was also found that, in case of mouse L cells, pretreatment with IFN (0t/13) in a concentration of 1000 U / m L inhibits the multiplication of L. pneumophi la (Dowling-Whitaker et al. 1986). Recently it was shown that Legionellte could not only infect WiREF cells, but also cause a specific cytopathic effect, viz. formation of plaques (Eberl-Gregori~ 1993).

In the present study the inhibitory effect of porcine interferon ~, (PolFN-),) on the replication of L. pneumophila in WiREF cells is described and this inhibition is compared with that caused by human interferons and some antibiotics.

M A T E R I A L S A N D M E T H O D S

Cells. Monolayer cultures of WiREF (Wistar rat embryonal fibroblasts) (Filipi~ et al. 1984; Cenci~ and Filipi~ 1993) cells were grown in Eagle's medium supplemented with 10 % foetal calf serum (FCS) at 37 ~ in 5 % CO2 atmosphere. The day before the experiments with Legionella were per- formed, the medium was used without antibiotics.

Interferons. The following interferons (IFNs) were used in the experiments: PolFN-y (Filipi~ et al. 1993, 1994) in a concentration of 104 U of antiviral activity tested on MDBK cells. The IFN used had a specific activity 105 U/rag protein. HulFN-), (Sigma, St. Louis, USA), 103 U / m L (specific activity 105 U/rag protein), HulFN-0t (Institute for Immunology, Zagreb, Croatia), 104 U of antiviral activity

*Corresponding author.

31 0 E. EBERL-GREGORI(~ et al. Voi. 41

per mL (specific activity 105 U/rag protein), rHulFN-cx (Alpha 2; Reaferon) (Protein, Moscow, Rus- sia), 104 U/mL (specific activity 106 U/rag protein), rHulFN-0~ (Alpha 1; Realdiron) (Protein, Moscow, Russia), 104 U /mL (specific activity 106 U/mg protein).

Bacteria. Two strains of Legionella pneumophila type 1 were used. One was a standard strain (designated as LP-G) and the other one was a wild strain (labeled as LP-L). The bacteria were grown on BCYE-Alpha agar. For the experiments the bacteria were resuspended in distilled water and adjusted to the requested turbidity according to McFarland. The number of bacteria according to McFarland was counted on BCYE-Alpha agar.

Infection and testing of IFNs. Experiments were performed on 2-d-old WiREF cells grown in microtiter plates. After a monolayer was formed, the medium without antibiotics was added to the cells. The cells were infected with the bacteria at a cell concentration of 3 x 108/mL. The effect of interferons was tested as follows: (a) the cells were infected and IFNs were added 1 d later; (b) the cells were treated with IFNs and 1 d later the bacteria were added; (c) IFNs and bacteria were added simultaneously. After 3 d the cells were fixed with glutaraldehyde and stained with methylene blue for 45 rain at 37 ~ Stained cells were washed with PBS (phosphate buffer saline, pH 7.3) and the bound color was extracted with a 0.1 mmol/L HCI. Absorbance was measured at 580 nm on a Dynatech spectrophotometer.

Electron microscopy. WiREF cells infected with Legionella (3 • 108 bacteria per mL) were fixed with a mixture of 1% of paraformaldehyde in 0.025 % of glutaraldehyde for 1 h and then with 1 % OSO4. After dehydration in an alcohol series the material was embedded'into the ERL (Spurr) for 1 h and overnight into the ERL. The polymerization took place at 70 ~ for 3 h. The thin sections were analyzed and photographed under the Jeol-JEM 1200 EX II electron microscope.

Statistical analysis. All experiments were performed 3 - 5 times in 8-10 parallels. The statisti- cal validity of each experiment was verified by the correlation coefficient (r); values of r were 0.78. Sig- nificance (p) values were calculated by a three-way analysis of variance.

RESULTS A N D DISCUSSION

Cytopathic effect of Legionella pneumophila. WiREF cells were infected with L. pneumophila in different inocula containing from 300 to 1 bacteria per cell. The number of plaques found depended on the number of bacteria (Fig. 1). A correlation was found between the number of bacteria and absorbance of the eluted color (Table I). Electron microscopic experiments revealed that infected after 1 d WiREF cells (Fig. 2) contained bacteria inside.

Table I. Correlation between the number of L. pneumo- phila cells per Wi R E F cell and absorbance at 580 nm

Number of bacteria Absorbance

per cell at 580 nm

300 0.50 • 0.07

150 0.61 • 0.05 75 0.67 • 0.09 38 0.81 • 0.11 19 0.87 • 0.15

9 0.89 • 0.19 5 0.93 • 0,05 3 0.95 • 0.06

1 0.93 • 0.08 0 0.96 • 0.03

Cell control 0.98 • 0.03

Table II. Effect of PolFN-y (104 U / m L ) on L. pneumophila

Combinations % of control

Cells + L. pneumophila 100 Cells + L. pneumophila + IFN a 93 Cells + (L. pneumophila) + IFN b 88 Cells + (IFN) + L. pneumophila c 50

aWiREF cells treated with PolFN-y and infected with

L. pneumophila at the same time. bWiREF cells infected with L. pneumophila 1 d before

PolFN-? treatment. CWiREF ceils pretreated with PolFN-y 1 d before L. pneu-

mophila infection.

Effect of PolFN-?. In experiments performed to study the effect of PoIFN-y on L. pneumo- phila, the cells were pretreated with 104 U/mL of PoIFN-y and then infected with L. pneumophila (Table II). An approximately 50 % inhibition was found. When the cells were treated with IFN simul- taneously with bacteria or a day after bacterial infection, there was no inhibition. Cell pretreatment

1996 PORCINE INTERFERON--/ INHIBITS THE GROWTH OF L. pneumoplu'la 311

with PolFN-,/led to the development of an antibacterial state resembling an antiviral state; The mech- anisms by which IFNs induce this state should be further elucidated. The possible direct antibacterial effect of PolFNq, in vitro was also tested. We could not establish any direct antibacterial effect resem- bling that of antibiotics (Eberl-Gregori/c. and Filipi~ 1995) (data not shown), although some authors (Nebozhina et al. 1991) claimed that IFNs affect the ultrastructural morphology of bacteria by causing damage to the cell wall, plasma membrane and ribosomal apparatus.

Fig. I. Cytopathogenic effect ofL. pneumophila in WiREF cells (arrows are pointing to the plaques).

Table IlL Comparison of antibacterial activity of different interferons

Interferon Concentration Growth U/mL inhibition a, %

HulFN-u 104 85_5 Reaferon 104 41.8 Realdiron 104 32_5 HulFN-7 103 29.4

aCalculated according to the formula:

% = ( A u c - AIFN)/AIc • 100,

where AUC is absorbance of untreated cells, AIFN absorbance of IFN-pretreated cells and AIC absorbance of infected cells (Nash eta/. 1988).

Table IV. Antibacterial activity of antibiotics a

Antibiotic Concentration Growth lag/L inhibition, %

Midekamycin 500 86.0 Rifampicin 10 62.4 Erythromycin 1000 22.6

alnhibition was calculated as in Table III.

Effect of human IFNs. To compare the antibacterial effect of PolFN-y the following human interferons were used: HulFN-~, -y, Realdiron and Reaferon. The results show differences in the per- centage of growth inhibition of infected cells (Table III).

312 E. EBERL-GREGORI(~ et al. Vol. 41

Fig. 2. Electron microscopic examination of WiREF cells infected with L. pneumophila; the bacteria can be seen in the celt cytoplasm (top; magnification 4 000x) and in the vacuoles (bottom; magnification 40 000x).

Effect of antibiotics. Some antibiotics were used in control experiments (Table IV). The results show differences in their effectiveness.

Combination of antibiotics and interferons. The effects of a combination of HulFN-tx with anti- biotics and PolFN-y with antibiotics were measured. The results show (Table V) that antibiotics were not effective in enhancing the antibacterial activity of the interferons. It seems that intraceUular bacte- ria are highly resistant to the bactericidal effects of both (Bhardway and Horwitz 1988) IFN and anti- biotics, alone or in combination.

1996 PORCINE INTERFERON-y INHIBITS THE GROWTH OF L. pneurnoplu'la 31 3

Interferon y (IFN-y) is a potent in vitro acti- vator of antimicrobial activity. In the case of human mononuclear phagocytes, an exposure to IFN-y causes restriction of intracellular growth of L. pneumophila (Gebran et al. 1994). When cells other than phago- cytes (fibroblasts or epithelial cells) were tested, IFN-g proved to be a better inducer of antibacterial activity. Experiments performed on epithelial Hep-2 cells (Nebozhina et al. 1991) showed that HulFN-0t decreased the adhesive capacity of bacteria and prob- ably inhibited the "spin phagocytosis". Comparison of HulFN-~ and -y with PolFN-y showed that the activity of HuIFN-cx is higher than that of HuIFN-y. This can be explained by the origin and type of WiREF cells. PoIFN-y shows an activity similar to that of HuIFN-e~. Our previous experiments pointed out the similarity between HuIFN-o~ and PoIFN-y (Filipi~ et al. 1994). The ability of IFNs to activate antibacterial mecha- nisms of cells other than professional phagocytes is not well characterized, although some reports sug- gested an important role of lymphokine-induced

Table V. Comparison of cell growth inhibition (%) caused by L. pneurnophila infection after a 1-d pre- treatment with a combination of interferons and antibiotics a

Ratio b ERY MID RIF

HulFN-~

1 : 1 66.3 55.8 59.1 2 : 1 81.0 70.8 67.0 1 : 2 76.3 69.0 70.2

PolFN-y

1 : 1 31.7 42.9 36.0 2 : 1 45.2 44.1 40.5 1 : 2 32.2 44.5 43.8

aCalculation as in Table III. blFN-to-antibiotic.

radicals in the defense against a variety of bacterial pathogens. The role of nitric oxide (NO) radicals in killing the intracellular pathogen L. pneumophila was examined in murine (RAW 264.7) and human (HL-60) cell monolayers pretreated with 100 U/mL of IFN-y (Summersgill et al. 1992). In the mouse system, NO plays a more critical role in the bactericidal activity than in the human system, where the absence of NO production limits IFN-treated HL-60 cells to bacteriostasis.

Because the identification of specific lymphokines with antibacterial activity has been lacking, our future experiments will be focused on the study of the possible mechanisms through which the PolFN-,/-induced antibacterial state in WiREF cells takes place. All though such inhibitory effects may depend mostly on the immunomodulatory role of IFN, in the case of L. pneumophila, like with some other members of Enterobacteriaceae (e.g. Salmonellae or E. coli), the possibility cannot be excluded that the inhibition may be due, at least in part, to direct inhibition of intracellular growth (Filchakov et al. 1994; Maleeva et al. 1988; Moulton and Gerner 1986) by IFN itself.

This work was supported by grants of the Slovenian Ministry for Science and Technology (research field: Biochemistry with Molecular Biology:. P1-5064-0381-95; J1-7437-0381-96).

REFERENCES

BHARDWAY N., HoRwrrz A.M.: Interferon gamma and antibiotics fail to act synergistically to kill Legionella pneumophila in human monocytes. ZIFN Res. g, 283-293 (1988).

CF_~ci~ A., l~tamg: B.: Biological activity of rat fibroblast interferon. Radiol.Oncol. 27, 307-311 (1993). DAISY J.A., BENSON C.E., McKrrRICK J., HAILPERIN M.C., TOMASSlr~I N., F'mEDMAN H.M.: Growth of Legionellapneuraophila in

tissue culture. Zlnfect.Dis. 143, 460-464 (1981). DOWUNG-WHrrAKER P., DOWLING J.N., LIU L., YOUNGER J.: Interferon inhibits the growth of Legionella micdadei in mouse

L-cells. ZIFNRes. 6, 107-114 (1986). EBERL-GRF_C, Om~ E.: Effect of human and human-like interferons on the bacterium Legionella pneumophila. (In Slovenian)

MSc Thesis. Medical Facuity-Biotechnical Faculty, University of Ljubljana 1993. EBERL-GREC, Om~ E., FIumd B.: The influence of human alpha interferon on cell infection with the bacterium Legionella

pneumophila. (In Slovenian) Zdrav.Vesm. 64 (III), 39-42 (1995). FERNA/qDEZ C.C., LEE S.H.S., HALDANE D., SUMARAH IL, ROZEE K.R.: Plaque assay for virulent Legionella pneumophila. Z Clin.

MicrobioL 27, 1961-1964 (1989). FILCHAKOV I.V., FILcamcov F.V., SPrVZa~ N.I.A., P R ~ E.I., IVANF~KO V.K.: Gamma-interferon and the antibacterial activity

of mouse macrophages in a Sa/mone//a infection. (In Russian) Zh.Mikrobiol.EpidemioLlmmunobiol. 1, 64-69 (1994). FILIPIC B., GOLOB A., STRUNA T., LIKAR M.: Porcine mitogen-induced interferon. Acta Virol. 38, 71-75 (1994). FlIaPi~ B., ROZMAN S., CARS.SON K., CF~Clg: A.: Natural porcine interferon gamma (PolFN-gamma). Radiol.Oncol. 27, 307-311

(1993). FHam~ B., R o ~ S., ~ r q K., CE~CI~ A.: Porcine interferon gamma (PolFN gamma). Zdrav.Vestn. 63 (II), 31-34

(1994).

$1 4 E. EBERL-GREGORI(~ et al. Vol. 41

FILIPI~ B., SCHAUER P., SUnAR A.: Changes of the intracellular levels of some hydrolases during spontaneus transformation of rat embryonal fibroblasts (REF) (Wistar strain). Farm.Vestn. 35, 239-243 (1984).

FRASER D.W., TSAI T.F., OI~_~STFaN W., PAmar~ W.E., BEECHAM H.I., SHARRAR ILG., HARMS J., MALLISON G.F., MARTIN S.M., McDADE J.E., SHEPARD C.C., BRACIlMAN P.S.: Legionaire's disease: Description of an epidemic of pneumonia. N.Engl.l.Med. 297, 1189-1197 (1977).

GEnRAN SJ., NEWTON C., YAMAMOTO Y., WIDEN IL, KLFAr4 T.W., FRIEDMAN H.: Macrophage permissivness for Legionella pneumophila growth modulated by iron. Infect.Immun. 62, 564-568 (1994).

GOBER LL., FRmDMAN-KIEN A.E., HAVELL E.A., VILCEK J." Suppression of the intracellular growth of Shigella flexneri in cell culture by interferon preparations and polyinosinic-polycytidylic acid. Infect.Immun. 5, 370-376 (1972).

JACOBS R.G., LOCKSLEY R.M., WILSON C.B., HAAS J.E., KLEBANOFF S.J.: Interaction of primate alveolar macrophages and LegioneUa pneumophila. J. Clin.lnvest. 73, 1515-1523 (1984).

JAHIEL R.I., VILCEK J., NUSSENZWEIG R., VANDERBERG J.: Interferon inducers protect mice against Plasmodium bergheri malaria. Science 161, 804-806 (1968).

MALWCA L.I., SERGEEV V.V., KUZNETSOV V.P., KORBOV V.T.: Partial purification and various characteristics of an antibacterial factor in preparations of interferon. (In Russian) Antibiotiki i Khimioterapia 33, 630-639 (1988).

MOULTON 1LG., GERNER G.D.: Antibacterial activity of BCG-induced interferon-containing sera. Can.l.MicrobioL 32, 442-445 (1986).

N~s, W.T., LmBY M.D., HoRwrrz A.M.: IFN-gamma-activated human alveolar macrophages inhibit the intracellular multipli- cation ofLegioneUapneumoplula. J.hnmunoL 140, 3978-3981 (1988).

NEBOZHINA L.V., KLrFSUNOVA N.V., TARTAKOVSKI! I.S., BYKOVSKII A.N.: The interaction of Legionella pneumoplu'la with differ- ent types of cell cultures and the effect of interferon preparation on this process. (In Russian) Zh.Milvrob.Epidem. Immunobiol. 6, 14-18 (1991).

NEBOZHINA L.V., KLrrSuNovA N.V., TARTAKOVSKI! I.S., KUZNETSOV V.P., BYKOVSKI! A.F.: The effect of interferon preparations on the ultrastructural organization of Legionella penumopIgla. (In Russian) Zh.Mil~obioLEpidem.ImmunobioL 12, 7-10 (1991).

P,~SCULLE A.W., FEELEY J.C., GIBSO)q ILl., CORDES L.G., MYEROWrrz ILL., PATRON C.M., GORMAN G.W., CPmMACK C.L., EZZELL J.W., DOWLit~G J.N.: Pittsburg pneumonia agent: Direct isolation from human lung tissue. J.Infect.Dis. 141, 727-732 (1980).

PAVNE N.IL, HoRwrrz A.M. : Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J.Ea:p.Med. 166, 1377-1389 (1987).

RINALI~ C.R., PASCULLE A.W., MYEROWlTZ ILL., GRESS F.M., DOWLi)qG J.N.: Growth of Pittsburg pneumonia agent in animal cell cultures, lnfect.Immun. 33, 939-943 (1981).

SUMblF_g~ILL J.T., POWELL LA., BUSTER B.L., MILLER ILD., RAMmEZ J.A.: Killing of Legionella pneumophila by nitric oxide in gamma-interferon-activated macrophages. J.Leukoc.Biol. 52, 625-629 (1992).

Y.~IAMOTO Y., OKUBO S., KLFarq T., ONOZAKI K., SArro T., FRIEDMAN H.: Binding of LegioneUa pneurnophila to macrophages increases cellular cytokine mRNA. ln.fect.lmmun. 62, 3947-3956 (1994).