Vol. 26, No. 2

INFECTION AND IMMUNITY, Nov. 1979, p. 698-704 0019-9567/79/11-0698/07$02.00/0

Serospecificity and Opsonic Activity of Antisera to Legionella pneumophila W. JOHNSON,'` E. PESANTI,2 AND J. ELLIOTT' Departments of Microbiology' and Internal Medicine,2 University of Iowa, Iowa City, Iowa 52242

Received for publication 6 August 1979

A high-molecular-weight surface component (F-1 fraction) has been isolated from the four serogroups of Legionella pneumophila. Antibody raised against live organisms was found by microagglutination assay to be specific for the homologous serogroup. Agglutinating activity of antiserum was markedly diminished after absorption with the homologous, but not heterologous, F-1 fraction. In addition, it was found that L. pneumophila organisms were not interiorized by rat alveolar macrophages or mouse peritoneal macrophages in the absence of antiserum, whereas homologous antiserum effectively opsonized the organisms. The opsonizing activity of serogroup-specific antisera was eliminated by absorption of the antisera with the homologous, but not heterologous, F-1 fraction. These data indicate that the serogroup-specific antigen of L. pneumophila resides in the F-1 fraction and that antibody to the F-1 fraction is required for phagocytosis of L. pneumophila by mammalian phagocytes. The organism isolated from cases of Legionnaires disease has been tentatively classified as Legionella pneumophila (3). Even with strict adherence to recommended procedures (10), the isolation of this organism has often proven difficult. This has resulted in emphasis on serological tests in the diagnosis of Legionnaires disease. The direct immunofluorescent test has been used to detect organisms in tissue (4), and the indirect immunofluorescent test is currently the standard method for determining antibody titers to L. pneumophila (10). Microagglutination and micro-enzyme-linked immunosorbent assay tests have also been developed for detection of antibodies to L. pneumophila (6). Although the serological tests have proved valuable in clinical diagnosis and epidemiological studies of Legionnaires disease, there is no information concerning the role of the antibodies detected by these serological tests in host defense against infections caused by L. pneumophila. In addition, the use of serological tests for the identification of L. pneumophila has been further complicated by the recent finding that isolates of L. pneumophila can be divided into four distinct serogroups based on direct immunofluorescent assay tests (11). In a previous study (8) we reported the isolation of a fraction (F-1) from the Philadelphia 2 strain (serogroup 1) which was shown to be the major antigen detected by human convalescent serum in both the indirect immunofluorescent and microagglutination assays. In the present

study, we have extended these observations to show that the F-1 fraction contains the serospecific antigen of the four serogroups of L. pneumophila detected in the microagglutination assay. In addition, evidence is presented to show that the phagocytosis of L. pneumophila by macrophages occurs only in the presence of serogroup-specific antiserum. MATERIALS AND METHODS Organisms. Representative strains of L. pneumophila serogroup 1 (Philadelphia 2), serogroup 2 (Togus), serogroup 3 (Bloomington 2) and serogroup 4 (LA) were obtained from the Center for Disease Control, Atlanta, Ga. Media and growth conditions. L. pneumophila was grown on Mueller-Hinton agar supplemented with 1% hemoglobin and 1% IsoVitaleX (BBL Microbiology Systems) at 370C in 5% CO2. For studies of phagocytic uptake, the organisms were grown in F-G broth (7) containing 10 ,uCi of [methyl1-H]thymidine (Schwarz/ Mann) per ml in an atmosphere of air and 5% CO2 on a tilt table at 37°C. Bacteria were washed three times in phosphate-buffered saline (PBS) before addition to macrophage cultures. Antigen isolation. The isolation of the F-1 fractions from each of the four serotypes was performed as previously described (8). Briefly, the procedure involves washing the cells from the surface of the agar with saline and incubating the cell slurry at room temperature for 1 h. The cells are removed by centrifugation, and the supernatant fluid is filtered and lyophilized. The lyophilized material is resuspended in one-tenth the original volume of distilled water and dialyzed against distilled water. The dialyzed material 698

VOL. 26, 1979

ANTISERA TO LEGIONELLA PNEUMOPHILA

is applied to a Sepharose 6B column, and the peak eluting in the void volume is dialyzed against distilled water and lyophilized. Microagglutination test. Microagglutination tests were performed by the procedure of Farshy et al. (6). Microagglutination inhibition tests were performed by incubating 0.1 ml of stock antigen suspension containing 2 mg of antigen (dry weight) per ml with 0.1 ml of serum for 2 h at 370C and then overnight at 40C. The absorbed sera was centrifuged at 4,000 x g for 15 min. The serum was removed and absorbed again with the stock antigen preparation. Immunodiffusion. Ouchterlony double immunodiffusion was done in 1% agarose made up in 0.02 M phosphate buffer containing 3% polyethylene glycol. Three milliliters of melted agar was layered onto a clean microscope slide (15 by 75 mm), and wells were punched in the solidified agar using an LKB gel punch. Ten microliters of antiserum or antigen was added to the wells, and the slides were incubated at room temperature in a humidity chamber for 48 to 72 h. Preparation of rabbit antisera. Antisera to live cells were prepared by intramuscular immunization of rabbits with 106 cells of live L. pneumophila suspended in Freund complete adjuvant. The rabbits received a booster immunization at 3 weeks and were bled 2 weeks after the booster immunization. Antisera to the F-1 fractions were prepared by intramuscular immunization of rabbits with 500 ,tg of F-1 antigen suspended in Freund complete adjuvant. Serum was obtained from the rabbits 3 weeks after immunization. Phagocytosis. Unstimulated mouse peritoneal macrophages were obtained from CF1 mice by peritoneal lavage. Rat alveolar macrophages were obtained from Sprague-Dawley rats by bronchial lavage. Macrophages were cultured as monolayers in tissue culture medium consisting of medium 199, heat-inactivated newborn calf serum (20%, vol/vol), penicillin, and gentamicin. Macrophage cultures were incubated in an atmosphere of air and 5% CO2 for at least 48 h before challenge with organisms. In all of the phagocytosis experiments, the ratio of bacteria to macrophages was ca. 100:1. For studies of phagocytosis of representative strains of the four serogroups, tritium-labeled organisms were added to macrophage monolayers to which had already been added fresh medium (without antibiotics) containing various dilutions of antisera. Phagocytosis was terminated by rinsing the monolayers three times with warm PBS, after which the monolayers were incubated for an additional 15 min in PBS containing 0.25% trypsin (1:250, Difco Laboratories). After trypsinization, the monolayers were rinsed four times with warm PBS. This rinsing procedure was found sufficient to minimize adherence of antibody-coated organisms to glass and macrophage surfaces. Monolayers were digested with 0.5 ml of 0.1 N NaOH. Uptake of organisms was calculated from the uptake of radiolabel as determined by liquid scintillation counting in dioxane and Ominofluor (New England Nuclear Corp.), and the numbers of macrophages were estimated by quantitation of monolayer protein by the method of Lowry et al. (9). Electron microscopy. Macrophages were fixed in

699

glutaraldehyde, scraped from the flasks, and suspended in PBS (pH 7.2). Cell pellets were prepared in a hematocrit centrifuge (5), postfixed in 1% OS04 in 0.15 M phosphate buffer (pH 7.2), dehydrated in acetone, and embedded in an Epon-Araldite resin mixture. Thin sections were stained with lead citrate. RESULTS

Specificity of the microagglutination response. The specificity of the microagglutination response in rabbits immunized with live L. pneumophila is shown in Table 1. Each of the rabbits showed the highest microagglutination titer when the homologous serotype was used as antigen. Serum from rabbits immunized with serogroup 1 had microagglutination titers of 1: 1,024 with the homologous antigen, whereas no significant titer was observed when serogroups 2, 3, or 4 were used as antigens. Antisera from rabbits immunized with live cells of serogroups 2, 3, or 4 all had microagglutination titers of .1: 4,096 when assayed with the homologous antigen. However, some cross-reactions were observed with the highest cross-reacting titer (1: 256) occurring when antiserum from rabbits immunized with serogroup 2 organisms was tested with serogroup 3 antigen. Role of F-1 fraction as the serospecific antigen. The importance of the F-1 fraction as the serospecific antigen detected by the microagglutination test is shown in Table 2. Serum from rabbits immunized with the live serogroup 1 strain had microagglutination titers of 1:1,024. Absorption of this serum with the F-1 fraction isolated from serogroup 1 organisms reduced the microagglutination titer to 1:8, whereas absorption with F-1 fractions from the other three serogroups had no effect on the microagglutination titer. Similar results were observed with the sera obtained from rabbits immunized with the other three serogroups. Absorption of each of these sera with the F-1 fraction from the homologous serogroup resulted in a significant TABLE 1. Specificity of microagglutination test titers of sera from rabbits immunized with live L. pneumophila serogroups 1, 2, 3, or 4 Microagglutination titera of serum from rabbits immunized with'

Antigen Serogroup 1

Serogroup 2

Serogroup 3

Serogroup 4

8 4 32 1,024 Serogroup 1 2 '4,096 64 64 Serogroup 2 4 128 256 >4,096 Serogroup 3 32 4 128 24,096 Serogroup 4 a Titers expressed as reciprocal of dilution. b Preimmune titers were all 16 or less.

700

JOHNSON, PESANTI, AND ELLIOTT

reduction of the microagglutination titer, whereas absorption with the F-1 fraction from heterologous serogroups did not result in a significant reduction of the microagglutination titer. Immunodiffusion reactions of the F-1 fraction from each of the four serogroups with antiserum to live cells are shown in Fig. 1. The F-1 fraction isolated from serogroup 1 gave a single precipitin line when reacted with antiserum to the serogroup 1 strain of L. pneumophila (Fig. 1A). The 1 fraction isolated from serogroup 2 (Fig. 1B), serogroup 3 (Fig. 1C), and serogroup 4 (Fig. iD) also showed a single precipitin line with homologous antiserum. None of the F-1 fractions

INFECT. IMMUN.

showed precipitin lines with antisera to the heterologous serogroups. Phagocytosis. Although large numbers of organisms are present in the alveolar macrophages of individuals with infections caused by L. pneumophila (2), there is currently no information concerning the role of antibodies in the phagocytosis of L. pneumophila. Therefore, to determine whether antibody is required for the phagocytosis of L. pneumophila, studies on the uptake of tritium-labeled L. pneumophila by peritoneal and alveolar macrophages in the presence of antiserum to the homologous and heterologous serogroups were undertaken. In preliminary experiments, examination of macrophage monolayers stained with Giemsa stain TABLE 2. Effect of absorption with homologous and indicated that neither alveolar nor peritoneal heterologous F-i fractions on the macrophages were capable of ingesting L. pneumicroagglutination titer of antisera to live L. mophila in the presence of normal serum or pneumophila antiserum to heterologous serotypes. However, Serum abMicroagglutination titera of antiserum to: addition of homologous antiserum resulted in sorbed with Fsubstantial phagocytosis of organisms by both SeroSeroSeroSero1 fraction cell types. Electron micrographs of mouse perifrom: group 1 group 2 group 3 group 4 toneal macrophages were obtained to insure that Unabsorbed 8,192 2,048 1,024 4,096 the L. pneumophila cells were located inside of 8 8,192 2,048 Serogroup 1 4,096 the macrophages. These results are shown in 32 8,192 2,048 Serogroup 2 1,024 Fig. 2. Opsonization of serogroup 1 and sero128 1,024 Serogroup 3 4,096 1,024 2 strains occurred in the presence of hogroup 32 4,096 Serogroup 4 4,096 1,024 mologous antiserum (Fig. 2A and C). However, Titers expressed as reciprocal of dilution. in the presence of antiserum to the heterologous serogroup very little interiorization of L. pneumophila was observed (Fig. 2B and D). To quantitate the uptake of L. pneumophila in the presence of homologous and heterologous antisera, each of the four serogroups of L. pneumophila were labeled with [3H]thymidine. The relative uptake ofradiolabeled organisms of each serogroup of L. pneumophila by peritoneal macrophages in the presence of homologous and heterologous antisera is shown in Table 3. For each of the four serogroups of L. pneumophila, maximum uptake of labeled bacteria by the peritoneal macrophages occurred when antisera to the homologous serogroup was present. Very little uptake occurred in the presence of antisera to heterologous serogroups. The importance of the F-1 fraction in determining the specificity of the opsonic activity of the antisera is shown in Table 4. Absorption of the antisera to each serogroup with the homologous F-1 fraction abolished the opsonic activity of the sera. However, FIG. 1. Immunodiffusion reactions of F-i fractions antisera absorbed with F-1 fraction isolated from with antisera to live cells of L. pneumophila. F-i heterologous serogroups retained their opsonic fractions from serogroup (Sl), serogroup 2 (S2), activity. In these experiments, then, homologous serogroup 3 (S3), and serogroup 4 (S4) are in the outside wells. Rabbit antisera to live (A) serogroup I antiserum effectively opsonized L. pneumophila, (ABi), (B) serogroup 2 (AB2), (C) serogroup 3 (AB3), while heterologous antiserum and antiserum aband (D) serogroup 4 (AB4) strains are in the center sorbed with homologous F-1 fraction were little, if any, more active in opsonization of L. pneuwells.

I_~~~~~~~~~~~~~~~~~~~~~~~~C

1

~

~

~

~

~

~

~

~

Au

IL

-V

D

Ah .Ses

..

*

I

.. ,

s

. ,

§

ri

E

tf .v.t

,,**,,'3

* \

..

.. P.

A. -.

..

'A

w\4

701

ANTISERA TO LEGIONELLA PNEUMOPHILA

VOL. 26, 1979

zA;

; s SS

2

#,S,- is

.o .

i:

. ,r

.; 31

v

-

*'.g .*'sS o-1zis

,

,.

*

7.

* :.

d

4-'* 1.

r *3

* eP5EFz

rA

_

" - "s

Serospecificity and opsonic activity of antisera to Legionella pneumophila.

Vol. 26, No. 2 INFECTION AND IMMUNITY, Nov. 1979, p. 698-704 0019-9567/79/11-0698/07$02.00/0 Serospecificity and Opsonic Activity of Antisera to Leg...
NAN Sizes 0 Downloads 0 Views