Istituto di Microbiologia, Facoltà di Medicina e Chirurgia, Seconda Università degli Studi di Napoli, Larghetto S. Aniello a Caponapoli n 2, 80138 Naples, Italy1
Author for correspondence: Marilena Galdiero. Tel: +39 081 5665662. Fax: +39 081 5665663. e-mail: marilena.galdiero{at}unina2.it
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ABSTRACT |
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Keywords: Salmonella typhimurium, lipopolysaccharide, cytokines, integrins, cellular receptors
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INTRODUCTION |
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ß2 leukocyte integrins (CD11a/CD18, CD11b/CD18 and CD11c/CD18) may also participate in LPS signalling (Ingalls & Golenboch, 1995 ; Ingalls et al., 1997
). This family of receptors are heterodimeric cell surface glycoproteins composed of a CD11 and a CD18 subunit. These integrins mediate cell adhesion to endothelial cell ligands such as intracellular adhesion molecules.
A number of studies have shown that porins from several Gram-negative bacteria also stimulate cells to produce and secrete cytokines (Galdiero et al., 1993 , 1995
, 2001
; Henderson et al., 1996
; Iovane et al., 1998
; Wilson et al., 1998
).
The precise mechanisms by which LPS activates cells have not yet been elucidated (Tabeta et al., 2000 ). One proposed model (Wright, 1995
) suggests that CD14 functions as an albumin-like carrier molecule that binds a large variety of molecules without recognition specificity, and that it can then transfer these molecules to unidentified recognition/cell-activating molecules in the cell membrane. According to this model, and considering the similarity between the biological effects of LPS and porins, and the utilization of CD14 and CD11a/18 as receptors also by components of Gram-positive bacteria, the aim of the present study was to test the hypothesis that CD14 receptors and CD11a/18 integrins may also function in signalling cytokine release following porin stimulation.
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METHODS |
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Porin preparation.
S. enterica serovar Typhimurium strain SH5014 was used to extract and purify porins. Porins were isolated from the lysozyme/EDTA envelopes as described by Nurminen (1985) . Briefly, 1 g of envelopes was suspended in 2% Triton X-100 in 0·01 Tris/HCl (pH 7·5, containing 10 mM EDTA); after the addition of trypsin (10 mg envelopes g-1), the pellet was dissolved in SDS buffer (4%, w/v, SDS in 0·1 M sodium phosphate, pH 7·2), and applied to an Ultragel ACA 34 column equilibrated with 0·25% SDS buffer. The fraction containing proteins, identified by A280, was extensively dialysed and checked by SDS-PAGE according to Laemmli (1970)
. The purity of the porin preparation was checked by SDS-PAGE, which revealed two bands, with molecular masses of 34 and 36 kDa. The protein content of the porin preparation was determined by the Lowry method and by the Limulus amoebocyte lysate assay (Limulus test) (Thye Yin et al., 1972
). The Limulus test showed the presence of LPS at 50 pg porins µg-1. The LPS concentration in the porin preparation was estimated to be <0·005% (w/w). In addition, polymyxin B (Sigma Aldrich) was incubated with porins to neutralize the biological activity of traces of LPS that could be present in the preparation. The porins were incubated with polymyxin B at room temperature for 1 h in a ratio of 1:10; LPS was incubated with polymyxin B in a ratio of 1:100 (Morrison & Jacobs, 1976
). LPS, porins and the polymyxin B mixture were used in pyrogen-free phosphate-buffered saline (PBS).
LPS preparation.
LPS-R was isolated from S. enterica serovar Typhimurium SH5014 with phenol/chloroform/petroleum ether as described by Galanos et al. (1969) . Briefly, liquid phenol (90 g dry phenol plus 11 ml water/chloroform/petroleum ether in a volume ratio of 2:5:8) was added to 1 g dried bacteria. After 2 min homogenization, bacteria were centrifuged and extracted twice. The supernatants were filtered through filter paper and treated as described by Galanos et al. (1969)
.
Cell line.
The human promyelomonocytic cell line THP-1 (ATCC TIB-202) (Tsuchiya et al., 1980 ) was cultured at 37 °C in a moist atmosphere of 5% CO2 in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U penicillin ml-1 and 100 µg streptomycin ml-1 (Labtek, Eurobio). To induce adherence to plastic and expression of CD14, cultures were grown in 6-well plates in the presence of 0·1 µM 1,25-dihydroxyvitamin D3 (vitamin D3) (Calbiochem-Novabiochem). After 72 h, cultures were fed by adding 0·5 vol. medium containing vitamin D3 and incubated for another 24 h (Kitchens et al., 1992
). In some serum-dependence experiments cells were washed four times with PBS and cultured in serum-free medium for 12 h before experimentation.
Western blot analysis of CD14 and CD11a protein in vitamin D3-treated and non-treated cells.
Cell pellets were resuspended in 50 µl lysis buffer containing 20 mM HEPES (pH 7·9), 25% (v/v) glycerol, 0·42 M NaCl, 12 mM MgCl2, 0·2 mM EDTA, 0·5 mM PMSF and 0·5 mM DTT. After three freezethaw cycles, cell lysates were centrifuged at 12000 g for 30 min, and the supernatant was saved. Samples (50 µg per lane) were separated by SDS-10% PAGE and transferred to nitrocellulose membrane. Anti-CD14 clone MEM-18, anti-CD14 clone UCHM-1 and anti-CD11a clone 38 mAbs were used. After several washes, the membrane was developed with an enhanced luminol reagent (Du-Pont, NEN) and exposed to Kodak X-Omat film.
Cell stimulation and cytokine release.
All assays were carried out using 3x106 THP-1 cells ml-1. Porins (505000 ng ml-1) and LPS (101000 ng ml-1) were prepared in pyrogen-free distilled water and then added to suspensions of THP-1 cells at a range of concentrations. These were then incubated for 24 h at 37 °C in an atmosphere of 5% CO2. After incubation the samples were centrifuged at 1800 r.p.m. at 4 °C for 10 min and the supernatants were collected and stored at -70 °C. All samples were assayed for the presence of cytokines (TNF-, IL-6 and IL-8) by ELISA, according to the manufacturers instructions (Roche Diagnostic). Cell viability was determined by measuring leakage of lactate dehydrogenase activity from cells into the supernatant, using a kit purchased from Roche Diagnostic.
Inhibition of CD14 and CD11a/18 binding.
Vitamin D3-treated THP-1 cells ( 4x106 cells ml-1) were incubated for 30 min at 4 °C with a range of dilutions of mAbs against CD14 or CD11a/18 (anti-CD14 clones MEM 18, isotype IgG, and UCHM-1, isotype IgG 2a; anti-CD11a clone 38, isotype IgG2a; and anti-CD18 clone MeM18, isotype IgG1) (Cymbus Biotechnology) and with appropriate non-immune isotype controls. This preparation was subsequently incubated with porins (505000 ng ml-1) or LPS (101000 ng ml-1). Cultures were incubated for 24 h at 37 °C in a humidified atmosphere (5% CO2 and 95% air). Vitamin D3-untreated cells were used to monitor CD11a/18 expression. In some experiments, we used as stimulus a mixture of porins plus polymyxin B. At the end of the incubation period, cells were centrifuged at 400 g at 4 °C for 10 min, and the supernatants were aliquoted and stored at -70 °C until they were assayed for the presence of TNF-, IL-6 and IL-8.
Antibodies.
Two mouse mAbs against human CD14 were used: MEM 18 (isotype IgG) and UCHM-1 (isotype IgG 2a). Anti-CD11a clone 38 isotype IgG2a and anti-CD18 clone MeM18 isotype IgG1 were utilized. Negative control immunoglobulins for the inhibitors experiments were purified mouse IgG1 and IgG2a (Cymbus Biotechnology).
Reproducibility of results.
The results are expressed as mean values±SD. The differences between the effects of anti-CD14, anti-CD11a/18 and control mAbs were analysed by the paired two-tailed sample t-test. The level of significance was set at 0·05.
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RESULTS |
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To determine whether traces of LPS were present, we carried out a Limulus test using as a standard a LPS solution which gave a Limulus test value of 0·1 EU ml-1. The LPS concentration in the porin preparation was estimated to be <0·005% (w/w). These trace amounts of LPS did not induce any biological activity under our experimental conditions (data not shown). The purity of the porin preparation from S. enterica serovar Typhimurium SH5014 was checked by SDS-PAGE. Coomassie blue staining revealed the presence of two protein species, with molecular masses of 34 and 36 kDa (Fig. 1a).
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CD14 protein expression correlates with vitamin D3 treatment of cells
To determine if the CD14 expression could also be detected by changes in protein levels, Western blot analysis was performed on both vitamin D3-treated cells and non-treated cell extracts. As indicated in Fig. 2, vitamin D3-treated cell extracts showed a strong cross-reactivity to anti-CD14 mAb MEM 18 and anti-CD14 mAb UCHM-1.
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To induce cell maturation and expression of surface CD14, THP-1 cells were pretreated with vitamin D3. Exposure to vitamin D3 induces adherence to plastic and expression of high levels of cell-surface CD14 (Fleit & Kobasiuk, 1991 ). More than 95% of adherent cells expressed CD14 as detected by mAbs (data not shown). Vitamin D3-treated THP-1 cells were incubated with S. enterica serovar Typhimurium porins or LPS. After 24 h, the production of TNF-
, IL-6 and IL-8 in the culture supernatants was determined by ELISA. The production of these cytokines was markedly higher in culture supernatants of THP-1 cells that had been stimulated with porins or LPS than in the unstimulated cells. Increasing the concentration of either porins or LPS led to increased levels of cytokine release (Fig. 3
). Cytokine release was evident with 50 ng porins ml-1 or 10 ng LPS ml-1; maximum production was achieved with 1000 ng LPS ml-1 or 5000 ng porins ml-1. In some experiments cytokine release was assayed under serum-free conditions. In this case, release of all three cytokines was induced by porins but not by LPS (data not shown).
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To examine whether cytokine production induced by porins is dependent on CD14, vitamin D3-treated THP-1 cells were preincubated with a range of concentrations of anti-CD14 mAbs, clone MEM 18 and clone UCHM-1, for 30 min at 4 °C.
After 24 h stimulation with porins (505000 ng ml-1) or LPS (101000 ng ml-1), supernatants were harvested, and TNF-, IL-6 and IL-8 activity was determined by ELISA (Table 1
). As expected, both anti-CD14 mAbs blocked the LPS-mediated production of TNF-
, IL-6 and IL-8 in a dose-dependent manner. Independently of their isotype (IgG or IgG 2a), the mAbs used showed inhibiting activity. Both anti-CD14 mAbs failed to inhibit cytokine production induced by porins at any of the dilutions tested. The anti-CD14 mAbs alone did not induce TNF-
, IL-6 and IL-8 production in THP-1 cells. The control mAbs which were used as isotype controls could not reduce the LPS-induced cytokine production. A non-specific stimulus, bovine serum albumin, did not induce any cytokine liberation (data not shown).
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In porin-treated cells, both anti-CD11a and anti-CD18 mAbs reduced the release of the three cytokines by about 2025%.
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DISCUSSION |
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During our studies we have also confirmed that the biological effect persists after treatment of porins with polymyxin B, while it is neutralized when LPS is treated with polymyxin B.
Our data show an involvement of CD11a/CD18 integrins in signal transmission in porin-stimulated cells. The inhibition of those receptors induces only a small decrease in cytokine release, which may, however, be biologically significant. It is possible that responsive cells may have other porin-binding sites.
Porin-specific receptors are still unknown. Therefore, it is possible that porin stimulation is not due to binding to specific receptors, but is a consequence of the perturbation of the cell membrane lipoproteic phase, induced during absorption or porin penetration (Galdiero et al., 1998 ; Tufano et al., 1984
). In vivo, during Gram-negative infection, porins and LPS do not act as single components, but act together as a lipoproteic complex (Hellman et al., 2000
; Vesy et al., 2000
).
Different Gram-negative bacteria have been shown to release several proteins that could act in combination with LPS to activate macrophages (Betz & Morrison, 1977 ; Galdiero et al., 1988
; Goldman et al., 1981
; Goodman & Sultzer, 1979a
, b
; Morrison et al., 1976
; Sultzer & Goodman, 1976
). Endotoxin protein preparations have been shown to contain at least 12 proteins, ranging in size from 5 to 80 kDa (Goldman et al., 1981
). The predominant proteins present in endotoxin preparations were the porins, protein II and lipoprotein (Goldman et al., 1981
), the most abundant protein in the outer membrane of bacteria in the Enterobacteriaceae (Evans & Pollack, 1993
; Sultzer & Goodman, 1976
). The LPS-associated protein complex can probably use different mechanisms of cell activation, which may or may not include specific receptors, as a consequence of the prevailing amounts of LPS or porins in such a complex. Whole bacterial cells, which express on their surface LPS and porins, use both CD14 (van Furth et al., 1999
) and CD11a/18 (Vazque-Torres & Ferric, 2000
) when interacting with leukocytes. Our results show that purified porins and LPS interact in different ways with the cell surface, both leading to cellular stimulation. Our data indicate that CD14 and CD11a/18 are involved in cytokine responses to LPS, but only CD11a/18 is involved in those to porins, and to a much lesser extent. Anti-CD14 mAbs and anti-CD11a/18 mAbs are capable of blocking LPS uptake by cells expressing these receptors and thereby prevent the LPS-induced release of proinflammatory cytokines. The same mAbs are not able to neutralize the biological activity of the porins.
In view of our findings, adjunctive treatment of severe Gram-negative bacterial infection, with antibodies to specific receptors, may not be straightforward.
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Received 21 March 2001;
revised 6 June 2001;
accepted 13 June 2001.