Effect of Pasteurella haemolytica outer membrane proteins on bovine neutrophils

FEMS Immunology and Medical Microbiology 20 (1998) 29^36

E¡ect of Pasteurella haemolytica outer membrane proteins on bovine neutrophils Giuseppe Iovane a; *, Massimiliano Galdiero a , Mariateresa Vitiello b , Luisa De Martino b a

Dipartimento di Patologia, Pro¢lassi ed Ispezione degli Alimenti, Sezione Malattie Infettive, Facoltaé di Veterinaria, Universitaé degli Studi di Napoli `Federico II', Via F. Delpino, 2-80137 Napoli, Italy b Istituto di Microbiologia, Facoltaé di Medicina e Chirurgia, Seconda Universitaé degli Studi di Napoli, Larghetto S. Aniello a Caponapoli, 2-80138 Napoli, Italy Received 16 July 1997; revised 7 November 1997; accepted 7 November 1997

Abstract The major outer membrane proteins (OMPs) isolated from Pasteurella haemolytica induce alterations of the biological activity of bovine polymorphonuclear leukocytes (PMNs). A dose-dependent reduction of the capacity of adherence to nylon wool in vitro was observed. OMPs also acted as chemotaxins at concentrations between 5 and 20 Wg/ml. Concentrations lower than 5 Wg/ml did not give considerable results. Preincubation with 5 Wg/ml of OMPs led to modifications in the values of the phagocytic index and of intracellular killing, which were found to be diminished with respect to controls. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Pasteurella haemolytica; Outer membrane protein (OMP); Bovine polymorphonuclear leukocyte (PMN)

1. Introduction Pasteurella haemolytica is the organism most commonly isolated from pneumonic lungs in feedlot ruminants and ruminants su¡ering from enzootic pneumonia, a disease resulting in signi¢cant economic losses to the beef and dairy industries. It is clear that no single factor is solely responsible for the occurrence of the disease; under conditions of immunodepression, as the number of damages accumulate and interact to create an environment in the respira-

* Corresponding author. Tel.: +39 (81) 440083; Fax: +39 (81) 458683.

tory tract of the animal that favour colonization and growth of a variety of bacterial agents, among others P. haemolytica. P. haemolytica possesses several components that may function as virulence mechanisms, e.g. lipopolysaccharide (LPS) [1^4], leukotoxin (LKT) [5^8], neuraminidase [9,10], capsular polysaccharide [11^13] and proteases [14^17] are known. Moreover, the role of outer membrane proteins (OMPs), as virulence factors, is not well known. Recently the ability to induce pulmonary in£ammation in vivo after bronchoscopic deposition of the proteinaceous component of endotoxin, LPS-associated protein (LAP), has been studied [18]. This preparation contains up to 12 proteins ranging from 10 to 35 kDa [19^21]

0928-8244 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 9 2 8 - 8 2 4 4 ( 9 7 ) 0 0 1 0 3 - X

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depending on the culture conditions or the method of extraction. In numerous previous investigations it has been shown that the endotoxin-associated protein fraction gives rise to many biological activities that interfere with the immune response. It a¡ects directly the macrophages-monocytes, polymorphonuclear neutrophils and lymphocytes by stimulating or inhibiting their biological activity like the release of cytokines and adhesion molecules, or by induction of the production of antibodies [18,22^24]. Besides LPS there are major OMPs among the structural components of the outer membrane of Gram-negative bacteria that stimulate antibody responses. The OMP pro¢les of Gram-negative bacteria are di¡erent, some proteins are porins and function in outer membrane permeability. These proteins isolated from Salmonella typhimurium and Helicobacter pylori have shown considerable endotoxic activity [25^29] like that of LPS, but probably with a di¡erent mechanism [30]. The porins are also able to induce in£ammatory processes [31] as shown for LAPs [18]. Furthermore, among the OMPs, the LPS-associated porins may represent the dominant antigenic factor which stimulates the production of protective antibodies [32,33]. The protective activity of OMPs used as antigen, has been demonstrated experimentally by using Neisseria [34] and Haemophilus [35] OMPs. It has also been shown that patients with pelvic infection and women with frequent gonococcal diseases present high levels of anti-OMP antibodies [34]. In the present study, the e¡ects of P. haemolytica OMPs on some bovine neutrophil biological activity in vitro were studied.

2.2. Preparation of OMPs

2. Materials and methods

Viable bovine polymorphonuclear leukocytes (PMNs) were obtained from heparinized venous blood of normal, healthy bovines using MonoPoly-Resolving-Medium (MPRM) (Flow Laboratories, UK). Contaminating erythrocytes were lysed with 0.155 M NH4 Cl. The isolated PMNs were washed three times in RPMI 1640 medium (Labtek Laboratories, Eurobio, Paris). Thirty minutes before the start of each assay, the neutrophils were exposed to di¡erent concentrations of OMPs at a tempera-

2.1. Bacteria and growth conditions P. haemolytica strain ATCC 14003 was used. Bacteria were grown in brain heart infusion (BHI) broth (Difco) for 18 h at 37³C under agitation; the cells were harvested at the end of the exponential growth phase and cell envelopes were prepared as described by Nikaido [36].

OMPs were isolated from the lysozyme-EDTA envelopes. Brie£y, 1 g of envelopes was suspended in 2% Triton X-100 in 0.01 M Tris-HCl (pH 7.5, containing 10 mM EDTA); after the addition of trypsin (10 mg/g of envelopes), the pellet was dissolved in sodium dodecyl sulfate bu¡er (SDS bu¡er, 4%, w/v, in 0.1 M sodium phosphate, pH 7.2), and applied to an Ultragel ACA34 column equilibrated with 0.25% SDS bu¡er. To obtain a leukotoxin-free preparation the samples were puri¢ed over an Amicon ultra¢ltration unit equipped with a 62-mm-diameter XM-50 ultra¢ltration membrane [37]. The fraction-containing proteins, identi¢ed by A280 , was extensively dialysed and checked by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) [38]. The protein content of the OMP preparation was determined by the method of Lowry et al. [39]. All possible traces of LPS were identi¢ed by gel-electrophoresis minislabs, stained with silver nitrate as described by Tsai and Frasch [40], and by the Limulus amoebocyte lysate assay [41]. The Limulus test showed the presence of LPS at 50 pg/Wg OMPs. The LPS concentration in the OMP preparation was estimated to be 6 0.005% w/w. In addition, OMPs were used in the presence of polymyxin-B (Sigma), at a ratio of 1:10, which neutralize the biological activity of traces of LPS that could be present in the preparation [42]. 2.3. Preparation of LPS LPS was isolated from P. haemolytica ATCC 14003 using the aqueous phenol procedure described by Westphal et al. [43]. 2.4. Polymorphonuclear leukocytes preparation

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31

ture of 37³C and washed three times in RPMI 1640 medium, only for the adherence assay this preincubation time was 1 h. Cell viability was evaluated with the trypan blue exclusion test.

where A = number of PMNs migrated towards chemoattractant site; B = number of random-migrated PMNs; C = maximum distance of chemotactic migration; D = distance of random migration.

2.5. Evaluation of PMN adherence to nylon wool

2.7. Phagocytosis and killing assays

PMN adherence was measured using the MacGregor technique [44]. Brie£y, 40 mg of nylon wool was packed to a height of 1.5 cm in a tuberculin syringe. Then 0.5 ml of PMN suspension in RPMI 1640 (5U106 PMN ml31 ) was allowed to percolate through the nylon column. The number of PMNs in the post-elution samples was determined by microscopic counts; the results were expressed as percentage of neutrophil adherence to the nylon. PMN adherence for a given specimen is de¢ned as the average value for three columns.

Phagocytosis capacity was evaluated in vitro on the interaction between PMNs and Staphylococcus epidermidis, according to the method of Van Zweet et al. [46]. Brie£y, 2U106 cells ml31 suspended in RPMI 1640 and 10% fetal serum were incubated with S. epidermidis (106 cells ml31 ). After 15, 30, 60 min a 0.5-ml sample was added to 1.5 ml of icecold RPMI 1640 to stop phagocytosis and centrifuged at 110Ug for 4 min. Two aliquots (0.1 ml) of three consecutive supernatant dilutions were plated for calculation of the number of bacteria. The phagocytic activity was expressed as the phagocytic index (Ft ) which is de¢ned as the decrease in the number of bacteria in the supernatant during a given interval and calculated according to the equation Ft = log N0 3log Nt , where N0 is the number of bacteria in the supernatant at the start and Nt is the number of bacteria in the supernatant at time t. Intracellular killing was done using the same scheme of treatment reported for phagocytosis following the technique of Van Zweet et al. [46]. Brie£y, after 15, 30 and 60 min phagocytosis of bacteria at 37³C, the noningested bacteria were removed by differential centrifugation (4 min at 110Ug) and washed twice at 4³C. Next, PMNs containing ingested bacteria were lysed by freezing the cell suspensions in liquid air (3170³C) and thawed at 37³C, three times. The number of viable intracellular bacteria was determined by the dilution plate technique. The level of intracellular killing at this time (t) was expressed as the percentage decrease in the number of viable intracellular bacteria according to the formula: log N0 3log Nt in which N0 is the number of viable intracellular bacteria at time 0 and Nt is the number of viable intracellular bacteria at time t.

2.6. Evaluation of PMN chemotaxis A modi¢ed method of migration under agarose of Nelson et al. [45] was used. Four ml of 1% agarose in RPMI 1640 medium were poured into 50-mm diameter Falcon Petri dishes. A treated or untreated PMN suspension (10 Wl) in RPMI 1640 (3U107 cells ml31 ) was placed in the center of three equally spaced wells cut into the agarose plate. An equivalent amount of zymosan-activated serum (5 mg ml31 ) or 1034 M N-formyl-L-methionyl-L-leucyl-Lphenylalanine (FMLP) or P. haemolytica OMPs at di¡erent concentrations with or without serum was placed into one of the lateral wells and served as a stimulant for chemotaxis. The other well was ¢lled with the appropriate non-chemotactic control medium (RPMI 1640). The agarose plates were incubated at 37³C in a humidi¢ed atmosphere containing 5% CO2 for 3 h in darkness, then ¢xed with methanol and 3.7% formaldehyde overnight at 4³C. After ¢xation the gel was removed intact and the plates were stained with the May-Grunwald Giemsa method and air-dried. Finally, the migrated cells were counted using an eyepiece grid under 40U magni¢cation and the migration index calculated using the following formula: Migration index ˆ

…A3B†U…C3D† 100

2.8. Statistics All experiments were carried out in triplicate; results were expressed as the mean þ S.D. Signi¢cance

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of di¡erence was determined by a Student's t-test, with statistical signi¢cance considered to be P 6 0.05.

3. Results 3.1. OMP preparations SDS-polyacrylamide gel electrophoresis (SDSPAGE) of OMP preparations is shown in Fig. 1. Electrophoretical analysis of the protein fraction extracted from P. haemolytica revealed three bands with molecular weights between 28 and 40 kDa. SDS-PAGE stained with silver nitrate [40] did not show appreciable amounts of LPS in the preparations. The Limulus test with a sensitivity-limit of 0.5 EU ml31 , turned out to be negative for the OMP solutions used in this study. The OMP-polymyxin B association did not modify the biological activity of the OMPs, while the LPS-polymyxin B complex was inactive [42]. 3.2. Adherence to nylon wool Preincubation of PMNs (5U106 cells ml31 ) with OMPs (0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 Wg ml31 ) for 1 h at 37³C, induced alterations on the physicochemical characteristics of the PMN surface. As shown in Table 1 the OMPs induced a reduction in Table 1 Inhibition of PMN adherence to nylon wool by treatment of the cells with P. haemolytica OMPs PMN treatment

% Adherence

PMNs in RPMI (control) PMNs preincubated with OMPsa 0.1 Wg 1.0 Wg 2.5 Wg 5.0 Wg 10.0 Wg 20.0 Wg PMNs preincubated with OMPs 1.0 Wg+PBb

94.4 þ 3.74 83.3 þ 2.05 72.2 þ 1.73* 66.7 þ 1.28* 55.6 þ 1.02* 50.4 þ 1.11* 48.2 þ 0.99* 74.0 þ 1.18*

The results represent the mean of three experiments þ S.D. a The PMNs (5U106 cells ml31 ) were incubated for 1 h at 37³C in RPMI 1640 medium containing OMPs at the concentration indicated above. b OMPs were incubated with polymyxin B (PB) at a ratio of 1:10 for 1 h at room temperature. An asterisk indicates statistical signi¢cance (P 6 0.05).

Fig. 1. Pattern of P. haemolytica OMPs on SDS-PAGE. Lane A: molecular weight standards (phosphorylase b, 94 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; K-lactalbumin, 14 kDa). Lanes B: di¡erent preparations of P. haemolytica OMPs (20 Wg).

adherence of PMNs to nylon wool, which is dosedependent, approximately 49% compared to control PMNs, when 20 Wg ml31 of P. haemolytica OMPs were used in the assay. The OMPs plus polymyxin B complex gave the same result as the OMPs alone. 3.3. Chemotaxis One of the more important responses of the phagocytes to in£ammatory stimuli is chemotaxis, de¢ned as an oriented cellular migration determined by a speci¢c chemical stimulus. The e¡ects of OMP-induced chemotaxis are reported in Table 2. The controls have been performed with zymosan-activated serum or with FMLP (1034 M). With zymosan-activated serum the PMN migration index was similar to

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G. Iovane et al. / FEMS Immunology and Medical Microbiology 20 (1998) 29^36 Table 2 Chemotaxis of PMN treated with OMPs or untreated PMNs

Chemoattractants

M.I.

Untreated

Zymosan-activated serum FMLP 1034 M OMPs-activated serum (5.0 Wg) OMPs-activated serum (20.0 Wg) OMPs 0.1 Wg OMPs 1.0 Wg OMPs 2.5 Wg OMPs 5.0 Wg OMPs 10.0 Wg OMPs 20.0 Wg LPS 5.0 Wg OMPs 20.0 Wg+PBa LPS 5.0 Wg+PBa OMPs 20.0 Wg+LPS 5 Wg+PBa

4.862 þ 0.402 4.982 þ 0.530 6.472 þ 0.720 5.743 þ 0.622 0.169 þ 0.203 0.988 þ 0.178 1.340 þ 0.220 1.831 þ 0.224 2.156 þ 0.314 2.669 þ 0.168 1.590 þ 0.194 2.250 þ 0.160 0.000 2.531 þ 0.158

Treated (OMPs 20.0 Wg)b Zymosan-activated serum FMLP 1034 M

0.000 0.000

The results expressed as migration index (M.I.), represent the mean of three experiments þ S.D. a The OMPs were incubated with polymyxin B (PB) at room temperature for 1 h at a ratio of 1:10, while LPS was incubated with polymyxin B at a ratio of 1:100. b PMNs (2U106 cells ml31 ) were incubated with OMPs (20 Wg ml31 ) for 30 min at 37³C.

that obtained with FMLP, while the value obtained with OMP-activated serum resulted in slightly higher values. OMPs also acted as chemotaxins at concentrations between 5 and 20 Wg ml31 ; lower concentrations had no signi¢cant results. Moreover, they exhibited a dose-dependent increase but in any case lower compared with control PMNs. A LPS concentration of 5 Wg ml31 determined a value of migration index similar to that obtained with 5 Wg ml31 OMPs. Polymyxin B neutralized the e¡ect of LPS chemotaxis, but not that of OMP Table 3 E¡ect of P. haemolytica OMPs on phagocytosis by bovine PMNs Time (min)

Ft Controls

0 15 30 60

0.0310 þ 0.003 0.2365 þ 0.040 0.2542 þ 0.050 1.1760 þ 0.200

chemotaxis. However, at con¢rmation of this the mixture OMPs, LPS and polymyxin B gave the same result as the OMPs alone. Preincubation of PMNs with 20 Wg ml31 of OMPs, for 30 min at 37³C, inhibited completely the migration induced by the chemotactic stimulus (serum plus zymosan or FMLP). 3.4. Phagocytosis and killing Preincubation of PMNs with P. haemolytica OMPs, at 37³C for 30 min, was able to modify both phagocytosis and intracellular killing. The results of the phagocytosis are reported in Table 3. In these experiments the PMN were treated only with 5 Wg ml31 , the lower active concentration. Lower concentrations gave results proportionally inferior while higher concentrations did not in£uence the process any more. The phagocytic index of cells preincubated with OMPs was found to be remarkably lower with respect to untreated PMNs. A lower phagocytic index of treated PMNs respect to untreated PMNs was evident already after 15 min of incubation with the bacteria. Untreated PMNs as well as PMNs treated with 5 Wg ml31 of P. haemolytica were also tested in killing assays (Table 4). A decrease of killing index of treated cells against untreated cells was also observed.

4. Discussion The results obtained show that P. haemolytica OMPs are a potent modulator of neutrophil activity. It is known that neutrophils migrate into the lung Table 4 E¡ect of P. haemolytica OMPs on the intracellular killing by bovine PMNs Time (min)

OMPs (5 Wg ml31 ) 0.0310 þ 0.003 0.1030 þ 0.035* 0.1122 þ 0.030* 0.1280 þ 0.047*

0 30 60

Data, expressed as phagocytic index (Ft ), are the mean of three experiments, each performed in duplicate, þ S.D. An asterisk indicates statistical signi¢cance (P 6 0.05).

33

Kt Controls

OMPs (5 Wg ml31 )

0.0128 þ 0.002 0.1176 þ 0.014 1.2890 þ 0.300

0.0128 þ 0.002 0.0163 þ 0.006* 0.0902 þ 0.003*

Data, expressed as killing index (Kt ), are the mean of three experiments, each performed in duplicate, þ S.D. An asterisk indicates statistical signi¢cance (P 6 0.05).

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within hours after the arrival of P. haemolytica [47,48]. As bacteria begin to multiply in the lung, they will release small amounts of outer membrane components among which LPS and OMPs by blebbing and by bacterial lysis. Our data suggest that these low levels of OMPs may stimulate chemotaxis and adherence of neutrophils favouring the heap in the initial in£ammatory focus. The OMPs of P. haemolytica inhibit phagocytosis and intracellular killing as previously shown by porins of S. typhimurium [26], favouring bacterial multiplication. On the other hand, P. haemolytica releases leukotoxin [5^8] which in turn at very low concentrations induces cytoskeletal alterations in bovine neutrophils, as measured by a signi¢cant shape change response and also stimulates the release of secondary granule constituents and oxygen intermediates [49]. The activation of neutrophils with the consequent release of surface constituents, OMPs and LPS [2], can damage lung cells. When continued bacterial multiplication occurs, increasing amounts of OMPs can be released which could consequently impair the defense functions of pulmonary neutrophils and in all probability also of other cells like the mononuclear phagocytes [50]. The OMPs may interact with the cells of the host organism and become either toxic or non-toxic depending upon their concentration [27]. In the case of Gram-negative bacteria, the OMP concentration is approximately 2U105 mol per cell and the lysis of about 107 cells is su¤cient for the release of OMPs in the range of amounts capable of biological activities. The importance of OMPs in infection pathogenesis can be con¢rmed in the literature by the potential role of antibody responses to outer membrane proteins in antibacterial protection. In recent years, the role of OMPs or porins as vaccines has been evaluated in numerous studies [51^56]. In the case of P. haemolytica infection with regard to experimental vaccine studies, calves vaccinated with live vaccines and bacterins in oil adjuvants had signi¢cantly greater resistance to challenge exposure than did nonvaccinated animals [57]; the presence of LKT-neutralizing antibodies, which are induced by live vaccines, was hypothesized to be an important factor in inducing resistance to pneumonic pasteurellosis [58,59]. Similar protection was shown

by bacterins in oil adjuvants in the absence of LKTneutralizing antibodies, indicating that antigens other than LKT are also important in protection [57]. This study demonstrates that P. haemolytica OMPs play a role in bovine neutrophils which is determinant in infection pathogenesis. Since the OMPs are certainly immunogens as shown for other Gram-negative bacteria, it is probable that also in P. haemolytica infection OMPs-neutralizing antibodies carry a protective function. In conclusion, P. haemolytica OMPs induce modi¢cation of characteristics of the bovine PMN surface and consequently of chemotaxis and phagocytosis, and are shown to be factors which interfere in pathogenicity mechanisms of such microorganisms.

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