An Outer Membrane Protein (Porin) as an Eliciting ... - Europe PMC

1 downloads 0 Views 1MB Size Report
hypersensitivity reaction consisting of mononuclear cell infiltration without polymorphonuclear cell ... often superimposed with the Arthus reaction, and histolog-.
INFECTION AND IMMUNITY, Mar. 1987, p. 822-824 0019-9567/87/030822-03$02.00/0 Copyright © 1987, American Society for Microbiology

Vol. 55, No. 3

NOTES An Outer Membrane Protein (Porin) as an Eliciting Antigen for Delayed-Type Hypersensitivity in Murine Salmonellosis Department

V. UDHAYAKUMARt AND V. R. MUTHUKKARUPPAN* School of Biological Sciences, Madurai Kamaraj University, Madurai-625 021, India

of Immunology,

Received 28 August 1985/Accepted 1 May 1986

The porin, an outer membrane protein of Salmonella typhimurium, was found to be a suitable antigen for eliciting delayed-type hypersensitivity in mouse salmonellosis. Histological examination of the reaction site revealed that the porin was superior to other antigenic preparations in eliciting a typical delayed-type hypersensitivity reaction consisting of mononuclear cell infiltration without polymorphonuclear cell contamination. This study indicates the importance of using a suitable protein antigen from S. typhi for human application.

The importance of cell-mediated immunity (CMI) in controlling Salmonella infections has been well documented (2, 3). Delayed-type hypersensitivity (DTH) is considered an in vivo manifestation of CMI, and development of the DTH reaction has often been correlated with protective immunity by early workers (3). Later studies have shown that DTH could not be correlated with protection in all situations (6, 9). It has been shown to vary among different mouse strains tested (6, 9). Recently, Killar and Eisenstein (6) found potection in the absence of DTH. In most of the studies, crude antigenic preparations of Salmonella containing a substantial level of lipopolysaccharide (LPS), such as heatkilled bacteria (1) and crude alcoholic extract enriched for proteins (3, 4), were used as eliciting agents. The trichloroacetic acid (TCA)-extracted cell wall preparation (8, 14) and ribosomal fractions (11) have also been used for DTH elicitation. The DTH reaction elicited by these antigens was often superimposed with the Arthus reaction, and histological examination revealed predominant infiltration of polymorphonuclear cells (PMN) in addition to mononuclear cells at the reaction site (4, 8). Collins and Mackaness (3) showed that it was necessary to have LPS-free protein antigens to elicit a typical DTH reaction. However, purified antigen is not used because it is difficult to purify. Furthermore, the use of crude bacterial extracts made it difficult to evaluate which component(s) elicited the antigenic activity. Recently, one group of workers (10) showed that in calves, 0 antigens presented as a macromolecular complex could be the epitopes that T cells recognize in anti-Salmonella DTH. Recently, the porin, a major outer membrane protein (OMP) from the cell wall of S. typhimurium, was purified and shown to be capable of inducing protective immunity and DTH in mice (7; unpublished data). In this study, we have examined the efficacy of porin in eliciting DTH in Salmonella-infected mice and compared it with that of OMPs and the TCA extract of S. typhimurium. The porin from S. typhimurium C5 was purified by the method of Tokunaga et al. (13). Sodium dodecyl sulfate-

A B C

*- 66K

*- 52K

K.-

43 K

_~~~2 K *

23K

FIG. 1. Electrophoretic analysis of porn from strain C5. Lanes: A, 15 ,ug of porn; B, 45 ,zg of porin; C, marker proteins. Molecular weights (103) are indicated.

polyacrylamide gel analysis showed that our preparation was pure, free of contamination with other proteins (Fig. 1). LPS was undetectable in this preparation when estimated by the carbocyanin dye method of Janda and Work (5). Furthermore, the identity of the purified porin was confirmed by testing with antiporin sera obtained from Prof. Nakae (Takai University School of Medicine, Japan) (Fig. 2). OMPs from S. typhimurium were prepared as described elsewhere (15). The TCA extract from S. typhimurium was prepared by the

*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. .. *::.....,,,.:

FIG. 2. Immunodiffusion pattern of porin with rabbit antiporin

sera (obtained from T. Nakae). Wells: 10 ,ul of antisera to porins of

Corresponding author. t Present address: Department of Medical Microbiology and Immunology, University of Kentucky, Lexington, KY 40536-0230. *

strains 5551 (well 1) and 6017 (well 2); 10 ,ug of C5 porin (well 3); normal rabbit serum (well 4). 822

NOTES

VOL. 55, 1987

TABLE 1. DTH response elicited by various antigenic preparations in S. typhimurium-infected mice Mean increase in footpad thickness (0.1-mm units) Elicitation (days postimmunization)

Immunization

Phosphate-buffered saline (control)

None 7 15 21 30

M525 (5 x 104)

a

Porin

after elicitation with: OMP

2.5 ± 0.3 7.4± 0.6 12 0.7 16 0.9 8.2 ± 0.4

7.5 ±0.5 11 0.7 14 0.5 9.2 ± 0.6

823

t

SEa TCA

extract

2.1 ± 0.2

0.9 ± 2.4 ± 6.1 ± 7.2 5.4 ±

0.1 0.3 0.4 0.7 0.4

Arithmetic mean for five to eight mice in each group.

method of Staub (12) as described by Padmanaban and Mittal (8). Groups of BALB/c mice were immunized by injecting 5 x 104 live S. typhimurium M525 intravenously as described elsewhere (14). M525 is a smooth strain with a 50% lethal dose (LD50) of 106 organisms for this strain of mice. It is capable of inducing both protective immunity and DTH (14). Control mice were treated similarly with phosphate-buffered saline. At weekly intervals DTH was tested by injecting 5 ,ug of the porin, OMP, or TCA extract into the hind footpad. The other hind footpad served as the buffer control. Footpad swelling was measured at different times postelicitation with a vernier caliper. The difference between the control and antigen-injected footpads constituted the increase in footpad thickness. When porin was used as the eliciting antigen, DTH was detected as early as 7 days after immunization, reached the maximum level by week 3, and persisted at significant levels at least up to week 4 (Table 1). This study shows that porin is a potent antigen for eliciting DTH in Salmonella-infected mice. In addition, the efficacy of porin in eliciting DTH was compared with that induced by the OMP and TCA extract of S. typhimurium. Porin was better than the TCA extract in eliciting a high level of response in terms of absolute increase

'A

& 40

'V

in footpad thickness (Table 1). In addition, porin was found to be as efficient as OMP in eliciting DTH. Histological studies were carried out to characterize the DTH reaction induced by porin and OMP. Mice immunized 21 days previously with live S. typhimurium were injected in the footpad with the eliciting antigen or with buffer. The degree of swelling was determined after 24 h, selected mice were killed, and the reaction site was fixed in Bouin solution and sectioned. Representative sections (6 ,um) stained with hematoxylin and eosin are shown in Fig. 3, 4, and 5. The absence of DTH reaction in the control footpad was evident (Fig. 3). The cellular infiltrate in the porin-injected footpads of immunized mice was predominantly mononuclear in nature, and PMN were rarely seen (Fig. 4). On the other hand, predominant infiltration of PMN in addition to mononuclear cells were observed in the OMP-injected mice (Fig. 5). Similarly, in mice injected with the TCA extract, the reaction site was shown to be infiltrated with both PMN and mononuclear cells (8). Significantly, among the three antigenic preparations, only porin was able to elicit a typical DTH reaction without inducing PMN infiltration. It is interesting to compare the present finding with the results of Collins and Mackaness (3), who showed that the protein antigenic fraction devoid of LPS was necessary to elicit a

.

i4io I

e DiA eo~~~~~~ '% 3* .

> s~~*~~ _^

*X, 0

: 't, t.

-p^

*

F

N-I.-

Opk*.

t F

w *4 " s '' b.

I-,b

4

.1

e

>

)ktl

4

~

-

FIG. 3. Histological section of the porin-injected footpad of control mice. High-power view showing the absence of leukocyte infiltration (magnification, x5OO).

v

e

v

~

~

~

~

O

-M.

FIG. 4. Histological section of the porin-elicited DTH reaction in the footpad of a live vaccinated mouse. High-power view showing predominant infiltration of mononuclear cells (magnification, x 500).

824

NOTES

INFECT. IMMUN.

4%lF

A~ ~

~

b1t> ~~;

94

4

e4 q8%s?

.t .o 4 4p

:rt

w

tio

tr

M.

:

FInlGht tHeseolfindigs ietisno themtn OM-lctodsugeTH thactio ch on t pamin fatn l PSe v cinO Pane d thue. extractwe beoin presponsibleforth infiltration of PMN atmtheireaction, site..

prisueior antigenic tother prepartions ih Pe intmnae eulciting typca Drth.srato,laigtoiflrto fP N Conslideringthese poninssibiities, weptngt suggest that pohn

could be usd as an eliciting antigen in mouse salmonellosis like purified protein derivative in mycobacterial infection. This may help to minimize the variability among the different

authors working on salmonellosis and possibly to draw a clearcut correlation between DTH and protection. It is also important to point out here that in human typhoid there is no suitable assay system at present to detect the CMI reaction in vivo or in vitro. It may be due to the lack of a suitable test

antigen

which

could

be

used

either

for

DTH

elicitation

without side effects or for proliferative response. The present study shows that

porin

does not cause the side effects

known to be induced by LPS (4). Therefore, it is worth considering the

porin from

S. typhi for human application as

well. In addition, it is significant that this protein was able to induce protective immunity in mice (7; unpublished data).

V.U. is grateful to the Council of Scientific and Industrial Re search (C.S.I.R.), Government of India, for the financial assistance in the form of SRF. This work was also supported by a research grant from C.S.I.R. to V.R.M. LITERATURE CITED 1. Cho, N., T. Saito-Taki, and M. Nakano. 1983. Delayed hypersensitivity in murine salmonellosis: specificity of footpad reaction in mice infected with rough mutants of Salmonella typhimurium. Microbiol. Immunol. 27:167-175. 2. Collins, F. M. 1974. Vaccines and cell-mediated immunity. Bacteriol. Rev. 38:371-402. 3. Collins, F. M., and G. B. Mackaness. 1968. Delayed hypersensitivity and Arthus reactivity in relation to host resistance in Salmonella-infected mice. J. Immunol. 101:830-845. 4. Hormaeche, C. E., M. C. Fahrenkrog, R. A. Pettifer, and J. Brock. 1981. Acquired immunity to Salmonella typhimurium and delayed (footpad) hypersensitivity in BALB/c mice. Immunology 43:547-554. 5. Janda, J., and E. Work. 1971. A colorimetric estimation of lipopolysaccharides. FEBS Lett. 16:343-345. 6. Kiliar, L. M., and T. K. Eisenstein. 1984. Differences in delayedtype hypersensitivity responses in various mouse strains in the C3H lineage infected with Salmonella typhimurium: strain SL 3235. J. Immunol. 133:1190-1196. 7. Kuusi, N., M. Nurminen, H. Saxen, and P. H. Makela. 1981. Immunization with major outer membrane protein (porin) preparations in experimental salmonellosis of mice: effect of lipopolysaccharide. Infect. Immun. 34:328-332. 8. Padmanaban, V. D., and K. R. Mittal. 1979. Cross-protection against Salmonella enteritidis infection in mice. III. Delayed hypersensitivity reaction and clearance of the challenge organism. Acta Microbiol. Acad. Sci. Hung. 26:293-300. 9. Plant, J., and A. A. Glynn. 1976. Genetics of resistance to infection with Salmonella typhimurium in mice. J. Infect. Dis. 133:72-78. 10. Robertsson, J. A., S. B. Svenson, and A. A. Lindberg. 1981. Salmonella typhimurium infection in calves: delayed specific skin reactions directed against the 0-antigenic polysaccharide chain. Infect. Immun. 37:737-748. 11. Smith, R. A., and N. J. Bigley. 1972. Detection of delayed hypersensitivity in mice injected with ribonucleic acid-protein fraction of Salmonella typhimurium. Infect. Immun. 6:384389. 12. Staub, A. R. 1967. Preparation of cell wall antigens from gram-negative bacteria, p. 28-34. In C. A. Williams and M. W. Chase (ed.), Methods in immunology and immunochemistry. Academic Press, Inc., New York. 13. Tokunaga, M., H. Tokunaga, Y. Okajima, and T. Nakae. 1979. Characterization of porins from the outer membrane of Salmonella typhimurium. 2. Physical properties of the functional oligomeric aggregates. Eur. J. Biochem. 95:441-448. 14. Udhayakumar, V., and V. R. Muthukkaruppan. 1983. Characteristics of live vaccines in relation to delayed-type hypersensitivity and protective immunity in murine experimental salmonellosis. Immunol. Lett. 6:299-302. 15. Udhayakumar, V., and V. R. Muthukkaruppan. 1987. Protective immunity induced by outer membrane proteins of Salmonella typhimurium in mice. Infect. Immun. 55:816-821.