a Department of Public Health and Microbiology, Microbiology Division; and b Biomedical Science and Human Oncology, University of Turin, Turin, Italy
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Abstract |
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Introduction |
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As we reported previously that co-amoxiclav possesses beneficial properties which result in a great in-vitro enhancement of the phagocytic and microbicidal activities of human PMNs towards penicillin-resistant Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pneumoniae, 2 ,3 ,4 we examined the effect of co-amoxiclav on cytokine production by lipoplysaccharide (LPS)-stimulated human granulocytes in vitro.
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Materials and methods |
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A clinical strain of K. pneumoniae was used in this study: an inoculum of 2 x 10 7 cfu/mL was obtained by serial dilution of a logarithmic phase culture in fresh BHI broth (Unipath, Milan, Italy).
Antibiotic
Amoxycillin trihydrate and lithium clavulanate were kindly provided by SmithKline Beecham (Milan, Italy). Antibiotic susceptibility testing was performed by the standardized dilution method in Mueller-Hinton broth (Unipath) using a combination of amoxycillin-clavulanic acid as 2/1 ratio with an inoculum of 2 x 10 7 cfu/mL; the MIC for the K. pneumoniae was 16/8 mg/L.
Opsonization procedure
Serum from a pool of healthy volunteers was used. After the blood had been allowed to clot for 1 h at room temperature, the serum was collected by centrifugation for 20 min at 1100g, and stored at -70°C until use. Human pooled serum was used unheated (intact complement system). Klebsiellae were incubated at 37°C with 10% human pooled serum; opsonization was stopped and serum was removed by centrifugation at 2000g for 10 min; the bacteria were then resuspended in fresh medium to a final concentration of 2 x 10 7 cfu/mL, as confirmed by obtaining colony counts in triplicate. 5
Polymorphonuclear granulocytes
PMNs were separated from lithium heparinized venous blood obtained from normal volunteers using Ficoll-Paque (Pharmacia S.p.A., Milan, Italy). 2 Using trypan blue testing, the viability of PMNs was determined as greater than 95%. PMNs were suspended in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY, USA) with 10% fetal calf serum (FCS; 10 6 PMN/mL). They were placed in sterile plastic tubes and incubated at 37°C in a shaking water bath (150 rpm) before the addition of K. pneumoniae. The interval between PMN harvest and the start of experiments was less than 30 min.
Cytokine production in PMNs
Klebsiellae (2 x 10 7 cfu/mL) in RPMI 1640 (Gibco) with 10% FCS, containing 0.5 MIC of co-amoxiclav with LPS (50 ng/mL), 1 were added to PMNs (2 x 10 6 cells/mL) and incubated at 37°C in a shaking water bath for different times (1- 2- 3- 4 h). Antibiotic-free controls were included.
RNA preparation, cDNA synthesis and semiquantitative PCR
Total cellular RNA was extracted with an acid guanidinium thiocyanate- phenol- chloroform
mixture according to the method of Chomczynski & Sacchi
6 using RNAzol solution (Cinna/Biotec, Houston, TX,
USA). For the complete inhibition of proteases during extraction, the cell suspensions were
incubated with 0.025 mg/mL of protease inhibitor cocktail (Boehringer Mannheim, Germany).
The cDNA synthesis was performed by reverse transcription at 42°C for 45 min in 50
µL of reaction mixture containing 2 µg of total RNA using 0.5 µg
oligo(dT) primers, 1 mmol/L dNTPs (2'-deoxynucleotide-5'-triphosphate),
5 µL 10 x RT buffer (100 mmol/L Tris- HCl, pH 8.8, 500 mmol/L KCl and
1%Triton X-100), 5 mmol/L MgCl
2, 20 U RNAsin and 12.5 U avian Moloney virus (AMV)-RT. Five microlitres of
cDNA were amplified in the PCR buffer (10 mM Tris- HCl, pH 8.8, 50 mM KCl, 2.5 mM MgCl
2 and 1% Triton X-100) containing 0.2 mM of each deoxynucleotide
triphosphate,
2.5 U of AmpliTaq DNA polymerase (Perkin- Elmer, Norwalk, CT, USA) and 25 pmol of each
sense and antisense primer. The mixture was overlaid with mineral oil and amplified in a
Thermal Cycler (Cetus Corp., Emeryville, CA, USA) with PCR cycle conditions individual for
the different cytokines tested. The PCR products were electrophoresed in a 2% agarose gel
in
Tris- boric-acid- EDTA. Gels were stained with ethidium bromide and photographed. The PCR
primers for gene specific amplification of cDNA in the analysis of mRNA expression (IL-8,
TNF and IL-1ß) were purchased from Clontech (Palo Alto, CA, USA). As a
positive control of RT- PCR the ß
2-microglobulin (ß
2-m) was amplified.
7 To determine the percentage of T cells, B cells and
monocytes contaminating the total mononuclear cells, the cells- samples were stained with the
anti-CD3, anti-CD19, and anti-CD14 monoclonal antibodies (Becton Dickinson, Mountain View,
CA, USA) according to standard procedures, and analysed by FACScan flow cytometer. The
degree of purity of PMNs was more than 90%. No CD14 mRNA production in
LPS-stimulated
PMNs was detected. The percentage of monocytes contaminating the total mononuclear cell,
stained with the antiCD14 antibody and analysed by flow cytometric, was 0.52.
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Results and discussion |
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We reported previously that, among ß-lactam antibiotics, co-amoxiclav results in a great
enhancement of the activities of human PMNs in vitro towards penicillin-resistant K. pneumoniae, S. aureus and S. pneumoniae: in the presence of the
combination, all the bacteria, either serum unopsonized or opsonized, show an increased
susceptibility to both phagocytic and microbicidal activity of PMNs.
2
,3
,4
In this study we have investigated the influence of
co-amoxiclav upon the cytokine release by human PMNs, rather than the more traditional blood
monocytes, because in an infectious disease the PMNs represent the first cell type that
encounters
and interacts with the infectious agents, modulating the host immune response. The results
indicate that co-amoxiclav was able to exert different effects on IL-8, IL-1ß and
TNF- mRNA production by LPS-stimulated PMNs.
The addition of subinhibitory concentrations of co-amoxiclav to PMNs, stimulated with LPS for 1 h, promoted an increase in IL-8 mRNA expression especially when klebsiellae were present, whereas the addition of K. pneumoniae to PMNs without drug induced a signal comparable with that seen in PMNs alone (Figure). In the absence of klebsiellae, the co-amoxiclav still worked, showing IL-8 mRNA expression in comparison with both PMNs alone and PMNs with LPS (Figure).
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Furthermore, we have demonstrated that the effects of co-amoxiclav on IL-1ß mRNA
production were similar to those observed for IL-8 mRNA expression, although IL-1ß
was totally absent in PMNs stimulated with LPS (Figure). The
mechanism by which this absence
of signal occurs remains unclear. However, we can assume that IL-1ß was antagonized
by natural inhibitors, such as the IL-receptor antagonist (IL-1ra), which binds to receptor sites
located on the same target cell.
12 Further investigations will be required. We have
evaluated TNF- mRNA production by LPS-stimulated PMNs. The expression of
TNF-
mRNA was present only after the addition of co-amoxiclav and totally nonexistent
when the drug was absent. These data suggest that K. pneumoniae alone was not able to
induce TNF-
mRNA expression. In future, experiments with measurement by ELISA
should be performed to obtain further relevant and quantitative data. In conclusion, the results of
this study provide evidence that co-amoxiclav may modify the acute-phase inflammatory
responses through its effects on cytokine production by PMNs, making this combination more
suitable for the treatment of infections in immunocompromised patients.
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Acknowledgments |
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Notes |
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References |
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Received 9 July 1998; returned 11 August 1998; revised 28 August 1998; accepted 24 December 1998