Activation of Complement System by Porins Extracted from Salmonella ...

INFECTION AND IMMUNITY, Nov. 1984, p. 559-563

Vol. 46, No. 2

0019-9567/84/110559-05$02.00/0 Copyright C) 1984, American Society for Microbiology

Activation of Complement System by Porins Extracted from Salmonella typhimurium FRANCESCO GALDIERO,l* MARIA A. TUFANO,' LINDA SOMMESE,' ANTONIO FOLGORE,1 AND FRANCESCO TEDESCO2 Istituto di Microbiologia, I Facoltta di Medicina e Chiruirgia dell'Universita di Napoli, Larghetto S. Aniello a Caponapoli, 2, 80138 Napoli, I and Istitiuto di Patologia Generale, Universitei di Trieste, Fleming, 22, 34100 Trieste,2 Italia Received 9 April 1984/Accepted 24 July 1984

The effect of porins purified from Salmonella typhimurium on the complement system was investigated both in vitro and in vivo. Incubation of porins with either human or guinea pig serum resulted in the consumption of the total complement activity when an amount of porins ranging from 8 to 10 ,ug per 100 ,ld of serum was used. The activation of the complement system was temperature dependent, suggesting an active process rather than passive adsorption of the complement components by porins. In addition, the activation had a fast kinetic and proceeded mainly through the classical pathway. This conclusion is supported by the consumption of Cls and C4 in normal human serum treated with porins and also by the depletion of C3 activity in the Cls-deficient serum which was marked only when purified Cls was added to the serum before incubation with porins. Injection of 100 ,ug of porins into guinea pigs induced profound complement consumption at 6 h postinjection that persisted up to 12 h. We conclude from this study that porins can effectively contribute to complement activation and to subsequent biological events induced by gram-negative bacteria.

The ability of gram-negative bacteria to activate the complement system has essentially been attributed to the lipopolysaccharide (LPS) of the bacterial cell wall, which acts by a mechanism involving both the classical and the alternate pathways. Pillemer et al. (26) originally reported that endotoxins would interact with serum complement by an antibody-independent mechanism. Marcus et al. (16) demonstrated an LPS-initiated consumption of C3 in the absence of C2, and Dierich et al. (1) confirmed that LPS can act through the alternate pathway. Gewurz et al. (3) showed that LPS was able to induce marked depletion of the six terminal components and only slight consumption of the early components. More recently, Liang-Takasaki et al. (11, 12) have found that differences in LPS polysaccharide structure result in a variable degree of complement activation and this, in turn, may adversely affect the extent of bacterial virulence. Another region of LPS involved in complement activation is the lipid A, which triggers the complemnt sequence through the classical pathway as a result of a direct interaction with Clq in the absence of specific antibody (13, 18). In the present study, we show that the complement system can also be activated by an additional constituent of the cell wall of gram-negative bacteria, the porins, which we have previously reported to exert immunomodulatory effects both in vitro and in vivo (36a; M. A. Tufano, F. Rossano, and F. Galdiero, FEMS Symposium on Bacterial and Viral Inhibition and Modulation of Host Defence, Pisa, Italy, September 1982). Data will be reported indicating that complement activation by porins proceeds at a fast rate and involves primarily the classical pathway.

the method of Nurminen (24) and further purified by means of phenol extraction to remove LPS contaminations (4). These were in the order of 10 pg/10 pLg of porins in the final preparation as assessed by Limulus amoebocyte lysate assay (35) (Microbiological Associates, Walkersville, Md.). Controls included endotoxin-free water (lot no. 2243/3 Bieffe) and Westphal-extracted Escherichia coli 0111:B4 LPS (Difco) (37). In addition, chemical analysis did not show the presence of 2-keto-3-deoxyoctonate (25), indicating that lipid A may be present only in trace amounts, if at all. The protein content of the porin preparation was determined by the method of Lowry et al. (14), using bovine serum albumin as standard. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out according to Laemmli (10). All samples were prepared for electrophoresis by solubilization at 100°C for 5 min in the sample buffer, which contained 0.0625 M Tris-hydrochloride (pH 6.8), 2% sodium dodecyl sulfate, 5% ,-mercaptoethanol, 10% glycerol, and 0.001% bromophenol blue. Gels were stained with Coomassie blue. LPS was extracted from S. typhimurium SH5014 grown in nutrient broth (Difco) by the hot phenolwater procedure of Westphal and Jann (37) as reported by Kiley and Holt (8). In vivo experiments. A total amount of 100 pg of porins, dissolved in 1 ml of phosphate-saline buffer (PBS) at 70°C and sonicated in a bath Sonifier, was inoculated intraperitoneally into each of five guinea pigs, and blood samples were collected at 1, 6, and 12 h after the injection for complement

testing. Sera. Sera were obtained from human and guinea pig blood samples, which were allowed to clot at room temperature for 1 h and were kept in small portions in liquid nitrogen. A pool of sera from 20 blood donors served as the source of human complement. The Cls-deficient serum was obtained from a patient with mesangioproliferative glomerulonephritis who had normal levels of all the complement components except for Cls, which was undetectable either antigenically or hemolytically (34). An apparently healthy young boy was the source of the C3-deficient serum to be reported elsewhere. The serum contained the nephritic factor, which was

MATERIALS AND METHODS

Preparation of porins. A strain of Salmonella typhimurium SH5014 kindly provided by N. Nurminen (Central Public Health Laboratory, Helsinki, Finland) and grown in nutrient broth (Difco Laboratories, Detroit, Mich.) served as the source of porins. These were extracted from the bacteria by *

Corresponding author. 559

560

GALDIERO ET AL.

A

INFEC-T. IN1IJIN.

B 94 k

67 k

43 k

3C) k

of the sera was evaluated according to the method described by Lachmann and Hobart (9). Briefly, a mixture of 200 RI of Veronal-buffered saline contcaining various concentrations of serum and 50 [LI of EA (3 x 10( cells per ml) was incubated for 30 min at 37°C, and the amount of Iysis was measured lat 415 nm after the addition of 1 ml of ice-cold PBS-EDTA. The results were expressed as CH50 units per milliliter of serum. A similar procedure was followed tor the hemolytic titration of Cls. C4. and C3. except that the EA were suspended in a 1:10 dilution of Cls-deficient serum, a 1:40 dilution of C4deficient serum. and a 1:15 dilution ot C3-deficient serum. respectively. The hemolytic activity of the components trom CS to C9 was tested on intermediate EAC1-3(hu) prepared by incubating 1%/( EA with 1/10 volume of reagent human R3 for 70 s at 37°C and then adding Antrypol (Suramin. Bayer. Federal Republic of Germany) at 1 mg/ml (33). RESULTS Complement activation by porins. The purity of porins used in the present research was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis that revealed two bands of molecular weight 34.000 and 36.000 (34K and 36K) (Fig. 1). The sera tested by means of passive hemoagglutination did not show antibodies to S. tYphlimuriumn porins. The ability of porins to trigger the complement system tested by measuring the residual complement activity of serum samples incubated with various amounts of porins at 37°C for 30 min (Fig. 2). It can be seen that porins, although already effective at a concentration of 10 lg/ml, induced almost complete depletion of complement activity at 80 p.g/ml. The results of a similar experiment performed with purified LPS indicate that this had to be used at a concentration twice that of porins to create a comparable effect. The kinetic of complement consumption by porins investigated by incubating 100 pl of a 1:10 dilution of normal human serum (NHS) with 10 pLg of porins at 37°C and testing was

20 k FIG. 1. Protein patterns of porin preparations made from S. tvphimurium SH50)14. Lanes: A. SH5014. 20 p.g of protein in the sample: B. molecular weight standards (phosphorylise b. 94.000: albumin. 67.000: ovalbumin. 43.0t)(: carbonic anhydrase. 30.00t): trypsin inhibitor. 20.1t)0).

was

responsible for the depletion of C3 and the partial consumption of the late-acting components. The C4-deficient serum was obtained from a strain of guinea pig originated from the National Institutes of Health. Bethesda. Md. S. tvphirnuriiim antiporin antibodies were tested in the sera by means of passive hemagglutination. using sheep erythrocytes adsorbed with porins as described in a previous paper (Tufano et al.. in press). Briefly. the porins (5 l.g/ml in PBS) were adsorbed on sheep erythrocytes. suspended in PBS. incubated in 2% tannic acid in PBS, and resuspended in a 1% final concentration in PBS. One portion was put aside to be used as control cells. Buffers. Veronal-buffered saline (pH 7.4) containing 0.1% gelatin. 0.15 mM Ca-. and 0.5 mM Mg> was used for hemolytic complement assays. PBS containing EDTA (PBSEDTA). pH 7.4. was made with 0.15 M NaCl, 5 mM sodium phosphate. and 10 mM EDTA and was employed to block the lytic reaction at the end of the incubation time. Complement reagents. The reagent human R3 was prepared from human serum treated with yeast cells as described by Lachmann and Hobart (9). Cls was purified from human serum by the method of Sakai and Stroud (30). Cobra venom factor obtained in purified form from naja naja venom (Sigma Chemical Co.. St. Louis. Mo.) through DEAE chromatography and Sephadex G-200 gel filtration (9). For activation of the alternate pathway, cobra venom factor used at the concentration required to deplete the hemolytic activity of 100 VI of a 1:10 dilution of human serum on 100 p.1 of 2% sensitized sheep erythrocytes (EA). Hemolvtic assays. The total complement hemolytic activity

ZE

20-

IO -4

-a

-j

was

was

AMOUNT OF PORIHS 4S1| =1 of srurn) FIG. 2. Total complement activity of the serum after incubaition with various amounts of porins or with buffer as control.

VOL. 46, 1984

COMPLEMENT AND PORINS OF SALMONELLA TYPHIMURIUM

561

20r c0

T

30 -

Z_

I

LA._

15-, ccm J

12 t=E

E cm

20 _

a

0

!

L"

_

!

C

10

sn

10

1

3

5

7

9

TIME (min)

FIG. 3. Kinetic of complement consumption by porins. Symbols: 0, control; *, 100 ,ul of a 1:10 dilution of NHS with 10 ,ug of porins incubated at 4°C; A, 100 ,ul of 1:10 NHS with 10 ,ug of porins incubated at 37°C.

the complement activity on 20-pl samples withdrawn at various intervals. The results were referred to a control sample in which NHS was incubated with Veronal-buffered saline. A mixture of NHS and porins was kept in melting ice as a control for nonspecific adsorption of complement components. Nearly 30% of the complement activity was consumed by 1 min of incubation at 37°C, and complete depletion was achieved by 10 min, whereas no consumption was observed in the sample kept cold (Fig. 3). Activation of the classical pathway by porins. Having established that porins activate the complement sequence, we next analyzed the pathway involved. To this purpose, NHS was incubated with porins as described for the kinetic experiment, and the hemolytic activities of Cls, C4, C3, and C5 to 9 were assayed at the end of the 30-min incubation. The tested components were all depleted to various extents; the consumption ranged from 50 to 63% (Table 1). The depletion of Cls and C4 is definite proof that the classical pathway is activated by porins. Evaluation of the alternate pathway. To test whether the alternate pathway was also implicated in the complement activation by porins, 100 pL1 of a 1:10 dilution of Cis-deficient serum was incubated with 10 ,ug of porins, and the hemolytic activity of C3 was measured after 30 min of incubation at 37°C. Figure 4 shows that 35% of C3 was consumed under strict conditions of alternate pathway activation, and that the TABLE 1. Consumption of various complement components by porins CH50 U/ml of serum Complement Controls component Porinsa x 9.16 x 105 3.47 105 Cls 5.26 x 105 2.55 x 105 C4 16.7 x 103 6.7 x 103 C3 86.2 x 103 31.8 x 103 C5-9 A 100-pld volume of 1:10 NHS was incubated witb 10 pLg of porins for 30 a

min at 37°C.

c d a b e FIG. 4. Effect of porins on the activation of the complement through the alternate pathway, measured as residual C3 hemolytic activity by using C3-deficient serum as indicated in the text. (a) 100 ,ul of 1:10 Cls-deficient serum; (b) 100 p.1 of 1:10 Cls-deficient serum plus 1:10 purified Cls; (c) 100 ,ul of 1:10 Cls-deficient serum was incubated with 10 ,ug of porins at 37°C; (d) 100 ,ul of 1:10 Clsdeficient serum with 10 ,ug of porins and 1:10 Cis purified; (e) 100 ,ul of 1:10 Cls-deficient serum with CVF.

addition of Cls to the Cls-deficient serum, which allows the activation of the classical pathway, caused almost complete depletion of C3. The consumption of C3 induced by cobra venom factor indicates that the alternate pathway was functionally intact in the Cls-deficient serum. In vivo complement activation by porins. Preliminary in vitro experiments suggested that guinea pig complement can also be activated by porins in a dose-dependent manner. Thus, a significant inhibition of complement activity was obtained at a concentration of 10 pLg of porins per 100 p.l of serum. Porins proved equally effective in vivo since an amount of 100 p.g injected intraperitoneally was sufficient to deplete the serum complement activity (Fig. 5). This effect began to be seen at 6 h postinjection and persisted up to 12 h. DISCUSSION LPS has long been considered the major constituent of gram-negative bacteria responsible for complement activation (2, 3, 26). The results of our study clearly indicate that porin is an additional component of the bacterial cell wall capable of triggering the complement sequence. In this regard, porins appear to be at least as efficient as LPS since the amount of porins required to induce complement consumption in 100 pl. of serum, ca. 5 to 8 p.g, is lower than that of LPS for a similar effect, which usually ranges from 10 to 25 p.g (2). These data can also be taken as definite proof to rule out the possibility that LPS contributes considerably to complement activation by porins, since the contamination of our porin preparation by LPS was on the order of 10 pg of LPS per 10 p.g of porins. One major distinguishing feature of porins and LPS lies in their ability to trigger the complement sequence through

INFFCT. IMMUN.

GALDIERO ET AL.

562

20r r LAJl

Lfi

=

.-0

lo1

E

>-

b

c

5. Complement activity of serum obtained from guinea inoculated with S. trphiniuriimn porins. (a) Serum collected 1 h after the injection of 1(t( [g of porins (tb) serum collected 6 h after the injection of 1(t( Vg of porins: (c) serum collected 12 h after the

FIG.

a

pig

injection

of 100 Vg of

porins

and.

more

importantly. by the complete

con-

of C3 in the Cls-deficient serum incubated with porins only after the addition of purified Cls. T he rapid rate of complement inactivation by porins. already apparent by 1 min of incubation and proceeding to completion within 10 min is further evidence, though indirect, for the involvement of the classical pathway. which is usually faster than sumption

the alternate pathway. It should be pointed out. however, that 35% of the C3 hemolytic activity is consumed in the Cls-deficient serum incubated with porins. suggesting a moderate activation of the alternate pathway. The type of interaction of the porins with the complement system is not clarified by the present experiments. The characteristic hydrophobicity of the outer membrane proteins (4. 6) may play an important role in favoring the binding of the complement components to gram-negative bacteria. particularly to those in rough condition in which porins are more exposed on the surface. Whether the complement

This work was supported bv Progetto Finalizzato per il Contr-ollo delle Malattie dia Infezione from the Consiglio Nazionale delle Ricerche. Italy (no. 830)t)65352 and 830067852) and by a grant from Istituto per lI'nfanzia Burlo Garofalo.' Trieste. Italy. LITERATURE CITED 1. Dierich, M. P., D. Bitter Suermann, W. Konig, U. Hadding, C. Gplanos, and E. T. Rietschel. 1973. Analysis of bypass activation ot Cls by endotoxic LPS aind loss of this potency. Immunology

24:721-733. 2. Gewurz. H. 1972'. Alternate pathways to activation of the complement system. p. 56-88. In D. G. Ingram (ed.). Biological activities of complement. Kager. Basel. Switzerland. 3. Gewurz, H., H. S. Shin, and S. E. Mergenhagen. 1968. Interac-

tion of the complenient system with endotoxin lipopolysaccharides. Consumption of each of the six terminal complement components. J. Exp. Med. 128:1t)49-1057. 4. Hancock, R. E. W., and H. Nikaido. 1978. Outer membranes of gram-negative bacteria. XIX. Isolation from PNeulldo1onaosl (le5.

6. 7.

however,

is mediated by antibodies or by direct with Clq remains to be established. The results of our in vivo experiments suggest that porin is a potent activator of the complement system since as little as 100 p.g injected intraperitoneally into a guinea pig can be highly effective in depleting the serum complement activity.

activation,

ACKNOW'LEDGMIENITS

porins.

different pathways. Thus, it is well known that complement activation by LPS proceeds mainly through the alternate pathway (3. 26). although the classical pathway may also be involved to a limited extent (13. 18). In contrast. porins activate predominantly the classical pathway, as indicated by the marked depletion of Cls and C4 in the serum treated with

These data lend support to the hypothesis that porins can contribute to promote a local inflammatory reaction as result of complement activation by porins released or exposed externally on gram-negative bacteria. In this activity porins can cooperate with LPS. the biological effects of which are likely to be mediated. at least in part, by the complement system (2). Both the classic aind alternate pathways of complement activation are involved in the bacteriolytic activity exerted by blood serum on gram-negative bacteria. The classic pathway probably acts on sensitive strains, causing prompt lysis, whereas the alternate pathway acts on less-sensitive strains and Iysis is delayed (36). The mechanism of lysis is not clear: the complete structure of LPS and high levels of proteins in the outer membrane make gram-negative bacteria more resistant to Iysis by serum (5. 7. 15. 17. 20. 23. 27. 32). It is probable that the major membrane proteins and LPS activate complement by the same physicochemical and spatial conditions (18. 21. 22. 28. 29). Complement activation is more evident when LPS and porins are extracted from outer membranes. Preparations of LPS with diffrent polysaccharide contents activate the classical and alternate pathways differently (13. 18). Different complement-dependent effects on platelets have also been observed (19. 31). Morrison et al. (19) have demonstrated a critical role of the alternate pathway in endotoxininitiated complement-dependent rabbit platelet lysis. which represents the most deleterious pathological effects of endotoxin. From all of these factors we can evaluate with interest the considerable activation (35%lr) ot the alternate pathway by porins.

interaction of

porins

8.

rIwiio.sa PAO1 and use in reconstitution and definition of the permeability barrier. J. Btacteriol. 136:381-390. Hildebrandt, J. F., L. W. Maver, S. P. Wang, and T. M. Buchanan. 1978. aeisseria goorriou acquire a new principal outer-membrane protein when transformed to resistance to serum bactericidal activity. Infect. Immun. 20:267-273. Homma, J. Y. 1968. The protein moiety of the endotoxin of Pseudomonais aeruginosat. Z. Allg. Mikrobiol. 8:227-230. Inoue, K. 1972. Immune bacteriolytic and bactericidal reactions. p. 177-222. In J. B. G. Kwapinski (ed.). Research in immunochemistry and immunobiology. vol. 1. University Park Press. Baltimore. Md. Kilev, P., and S. C. Holt. 1980. Characterization of the lipopolysaccharide from Actinlohbaillus (t(ti/l01ZV((telfl(Olnita/IN Y4 and

N27. Infect. Immun. 30:862-873.

9. Lachmann, P. J., and M. J. Hobart. 1978. Complement technologv. chapter 5a. In 1). M. Weir (ed.). Handbook of experimental

VOL. 46. 1984

COMPLEMENT AND PORINS OF SALMONELLA TYPHIMURIUM

immunology. 3rd ed. Blackwell. Oxford. England. 10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 11. Liang-Takasaki, C. J., N. Grossman, and L. Leive. 1983. Salmonellae activate complement differentially via the alternative pathway depending on the structure of their lipopolysaccharide 0-antigen. J. Immunol. 130:1867-1870. 12. Liang-Takasaki, C. J., H. Saxon, P. H. Makela, and L. Lieve. 1983. Complement activation bv polvsaccharide of lipopolysaccharide: an important virulence determinant of salmonellae. Infect. Immun. 41:563-569. 13. Loss, M., D. Bitter-Suermann, and M. Dierich. 1974. Interaction of the first (CI). the second (C2) and the fourth (C4) component of complement with different preparations of bacterial lipopolysaccharides and with lipid A. J. Immunol. 112:935-940. 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275. 15. Madonna, G. S., and R. C. Allen. 1981. Slhiiella llonnei phase I and phase Il: susceptibility to direct serum lysis and opsonic requirements necessarv for stimulation of leukocyte redox metabolism and killing. Infect. lmmun. 32:153-159. 16. Marcus, R. L., H. S. Shin, and M. M. Maver. 1971. An alternative complement pathway: C3-cleaving activity not due to C4, 2a on endotoxic lipopolysaccharide after treatment with guinea pig serum; relation to properdin. Proc. Natl. Acad. Sci. U.S.A. 68:1351-1355. 17. Milner, K. C., J. A. Rudbach, and E. Ribi. 1971. General characteristic. p. 1-65. In G. Weinbaum. S. Kadis. and S. J. Ajl (ed.), Microbial toxins. IV. Bacterial endotoxins. Academic Press, Inc., New York. 18. Morrison, D. C., and L. F. Kline. 1977. Activation of the classical and properdin pathways of complement by lipopolysaccharide (LPS). J. lmmunol. 118:362-368. 19. Morrison, D. C., L. F. Kline, Z. G. Oades, and P. M. Henson. 1978. Mechanisms of lipopolysaccharide-initiated rabbit platelet responses: alternative complement pathway dependence of the lytic response. Infect. Immun. 20:744-751. 20. Munn, C. B., E. E. Ishiguro, W. W. Kay, and T. J. Trust. 1982. Role of surface components in serum resistance of virulent Aeromonas salmonicida. Infect. Immun. 36:1069-1075. 21. Muschel, L. H., and J. E. Jackson. 1966. The reactivity of serum against protoplasts and spheroplasts. J. Immunol. 97:46-51. 22. Muschel, L. H., and L. J. Larsen. 1970. The sensitivity of smooth and rough gram-negative bacteria to the immune bactericidal reaction. Proc. Soc. Exp. Biol. Med. 133:345-348. 23. Nelson, B. W., and R. J. Roantree. 1967. Analyses of lipopolysaccharides extracted from penicillin-resistant, serum sensitive

563

Salmonella mutants. J. Gen. Microbiol. 48:179-188. 24. Nurminen, N. 1978. A mild procedure to isolate the 34K. 35K and 36K porins of the outer membrane of Salmonella typhimurium. FEMS Microbiol. Lett. 3:331-334. 25. Osborn, M. J. 1963. Studies on the gram-negative cell wall. 1. Evidence for the role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S.A. 50:499-506. 26. Pillemer, L., M. D. Schoenberg, L. Blum, and L. Wurz. 1955. Properdin system and immunity. 11. Interaction of the properdin system with polysaccharide. Science 122:545-549. 27. Rowley, D. 1968. Sensitivity of rough gram-negative bacteria to the bactericidal action of serum. J. Bacteriol. 95:1647-1650. 28. Rowley, D. 1973. Antibacterial action of antibody and complement. J. Infect. Dis. 128(Suppl.):170-175. 29. Rowley, D., and K. J. Turner. 1968. Passive sensitization of Salmonella aldelaide to the bactericidal action of antibody and complement. Nature (London) 217:657-658. 30. Sakai, K., and R. M. Stroud. 1973. Purification, molecular properties and activation of Cl proesterase. Cls. J. Immunol. 110:1010-1020. 31. Siraganian, R. P. 1972. Platelet requirement in the interaction of the complement and clotting system. Nature (New' Biol.) 239:208-210. 32. Taylor, P. W. 1975. Genetical studies of serum resistance in Escherichia coli. J. Gen. Microbiol. 89:57-66. 33. Tedesco, F., M. Bardare, A. M. Giovanetti, and G. Sirchia. 1980. A familial dysfunction of the eight component of complement (C8). Clin. Immunol. Immunopathol. 16:180-191. 34. Tedesco, F., A. Tarantino, A. M. Givanetti, A. de Vecchi, C. M. Silvani, and E. Imbasciati. 1980. Deficiency of Cls: a case report. Fifth European Complement Workshop, Lund. Sweden. 35. Thye Yin, E., C. Galanos, S. Kinsky, R. A. Bradshaw, S. Wessler, 0. Luderity, and M. F. Sarmiento. 1972. Picogramsensitive assay for endotoxin: gelation of Limulus polyphemus blood cell lysate induced by purified lipopolysacharide and lipid A from gram-negative bacteria. Biochim. Biophys. Acta 261:284-289. 36. Traub, W. H., and I. Kleber. 1976. Selective activation of classical and alternative pathways of human complement by "promptly serum-sensitive' and 'delayed serum-sensitive strains of Serratia marcescens. Infect. Immun. 13.1343-1346. 36a.Tufano, M. A., M. T. Berlingieri, L. Sommese, and F. Galdiero. 1984. Immune response in mice and effects on cells by outer membrane porins from Sallmonella typhimrurimn. Microbiologica 7:353-366. 37. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharide extraction with phenol water and further applications of the procedures. Methods Carbohydr. Chem. 5:83-91.