Co-amoxiclav affects cytokine production by human polymorphonuclear cells

Gigliola Reatob, Anna Maria Cuffinia,*, Vivian Tullioa, Angela Ianni Palarchioa, Alessandro Boninoa, Roberto Foab and Nicola A. Carlonea

a Department of Public Health and Microbiology, Microbiology Division; and b Biomedical Science and Human Oncology, University of Turin, Turin, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Some antimicrobial agents have been reported to modify the host immune responses both in vivo and in vitro. As we demonstrated previously that co-amoxiclav had beneficial properties which result in enhancement of the microbicidal functions of human poly- morphonuclear cell (PMNs), we investigated the modulatory effect of this combination on cytokine production by human PMNs in vitro. The addition of co-amoxiclav elicited the production by lipopolysaccharide (LPS)-stimulated PMNs of substantial amounts of some cytokines, namely IL-8 and IL-1ß, after the addition of Klebsiella pneumoniae. These cytokine levels were higher than those obtained by PMNs incubated in culture medium only, without co-amoxiclav.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Polymorphonuclear cells (PMNs) usually represent the predominant cell type in an inflammatory response, acting as the first line of defence against invading bacteria. Recently, a growing body of evidence has demonstrated that PMNs have the ability to produce a number of immunomodulators, cytokines in particular, that may regulate the inflammatory cascade. 1 The cytokines function as essential mediators of cell-to-cell signals in physiological and pathological immune responses and represent alarm signals for the inflammatory response: the cytokines are rapidly produced in response to stimulation by infectious agents (or their derived products, inflammatory mediators or mechanical injury) and act as crucial signals in the development of appropriate defences. Among those, interleukin-8 (IL-8), tumour necrosis factor alpha (TNF-{alpha}) and IL-1ß have particular importance 1 and their potential production by PMNs therapeutically might prove to be beneficial in the treatment of bacterial infections, especially in severely immunocompromised hosts where infectious diseases represent a continuing threat. In the treatment of these infections the administered antimicrobial drugs often act together with host defences to eradicate the aetiological agents.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacteria

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{alpha} 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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Several antibiotics (fluoroquinolones, macrolides and some ß-lactam drugs), in addition to their antimicrobial effects, have the capability of modulating the host immune responses in different ways: 2 ,5 ,8 ,9 ,10 ,11 some are concentrated in the phagocytes where they enhance intracellular killing, whereas others become inert following intracellular uptake; some lead to alterations in bacteria that render them more susceptible to phagocytosis; some directly enhance the phagocytic and antimicrobial activities of the host cells; some depress the bacterial uptake by phagocytes; and others modulate T and B cell responses. Recent investigations have demonstrated that some antibiotics can also interfere with the cytokine production, some inhibiting the production of IL-1 or TNF-{alpha} in LPS-stimulated human monocyte cultures, others inducing hyperproduction of IL-2 and interferon gamma (IFN-{gamma}) in cultures of lymphocytes. 12,13

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-{alpha} 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).



View larger version (71K):
[in this window]
[in a new window]
 
Figure. (a) ß 2-microglobulin, (b) TNF{alpha}, (c) IL-8 and (d) IL-1 mRNA levels in lipopolysaccharide (LPS)-stimulated polymorphonuclear cells (PMN) incubated with 0.5 MIC of co-amoxiclav (AUG) and K. pneumoniae (Kleb.).

 
No significant differences were detectable with different LPS stimulation times (2- 3- 4 h). A similar pattern was observed by adding either heat-killed bacteria or LPS alone. Moreover, serum pre-opsonization of K. pneumoniae had no significant effect on cytokine release in comparison with that observed with non-opsonized bacteria (data not shown).

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-{alpha} mRNA production by LPS-stimulated PMNs. The expression of TNF-{alpha} 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-{alpha} 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.


    Acknowledgments
 
This study was supported by grants from the Italian MURST and AIRC (Associazione Italiana per la Ricerca sul Cancro).


    Notes
 
* Corresponding author. Department of Public Health and Microbiology, Microbiology Division, Via Santena 9, 10126 Torino, Italy. Tel: +39-11-670.6613; Fax: +39-11-66.36.436; E-mail: cuffini{at}molinette.unito.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Derevianko, A., D'Amico, R. & Simms, H. (1996). Polymorphonuclear leucocyte (PMN)-derived inflammatory cytokines regulation by oxygen tension and extracellular matrix. Clinical and Experimental Immunology 106 , 560 –7[ISI][Medline]

2 . Cuffini, A. M., Tullio, V., Paizis, G. & Carlone, N. A. (1996). Potential effects of amoxycillin/clavulanic acid and ticarcillin/clavulanic acid on human granulocyte activity: a comparative study. Journal of Antimicrobial Chemotherapy 38 , 1013 –22[Abstract]

3 . Cuffini, A. M., Tullio, V., Allocco, A., Paizis, G. & Carlone, N. A. (1996). The antibacterial activity of amoxycillin/clavulanic acid against Staphylococcus aureus ingested by human granulocytes. Microbios 87 , 31 –8[ISI][Medline]

4 . Cuffini, A. M., Tullio, V., Ianni Palarchio, A., Bonino, A., Paizis, G. & Carlone, N. A. (1998). Enhanced effects of amoxycillin/clavulanic acid compared to amoxycillin and clavulanic acid alone on the susceptibility to immunodefences of a penicillin-resistant strain of Streptococcus pneumoniae. Drugs Experimental Clinical Research24 , 9 –15

5 . Cuffini, A. M., Tullio, V., Bonino, A., Allocco, A., Ianni Palarchio, A. & Carlone, N. A. (1998). Entry of sanfetrinem into human polymorphonuclear granulocytes and its cell-associated activity against intracellular, penicillin-resistant Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42 , 1745 –50[Abstract/Free Full Text]

6 . Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol- chloroform extraction. Analytical Biochemistry 162 ,156 –9[ISI][Medline]

7 . Suggs, S. V., Wallace, R. B., Hirose, T., Kawashima, E. H. & Itakura, K. (1981). Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human ß 2 microglobulin. Proceedings of the National Academy of Sciences of the USA 78 , 6613 –7[Abstract]

8 . Cuffini, A. M., Carlone, N. A., Tullio, V. & Cavallo, G. (1993). Cell wall inhibitors and bacterial susceptibility to phagocytosis. In Host Defences Dysfunction in Trauma, Shock and Sepsis (Faist, E., Meakins, I. L. & Schildberg, F. W., Eds), pp. 979–85 Springer- Verlag, Berlin.

9 . Gemmell, C. G. (1993). Antibiotics and neutrophil function: potential immunomodulating activities. Journal of Antimicrobial Chemotherapy 31, Suppl. B , 23 –33[ISI][Medline]

10 . Labro, M. T., el Benna, J. & Abdelghaffar, H. (1993). Modulation of human polymorphonuclear neutrophil function by macrolides: preliminary data concerning dirithromycin. Journal of Antimicrobial Chemotherapy 31, Suppl. C , 51 –64[ISI][Medline]

11 . Cuffini, A. M., Tullio, V., Allocco, A., Paizis, G., DeLeo, C. & Carlone, N. A. (1994). Effect of rufloxacin upon non-specific immune-defences: in-vitro, ex-vivo and in-vivo results. Journal of Antimicrobial Chemotherapy 34 , 545 –53[Abstract]

12 . Morikawa, K., Watabe, H., Araake, M. & Morikawa, S. (1996). Modulatory effect of antibiotics on cytokine production by human monocytes in vitro. Antimicrobial Agents and Chemotherapy40 , 1366 –70[Abstract]

13 . Riesbeck, K. & Forsgren, A. (1995). CP-115,953 stimulates cytokine production by lymphocytes. Antimicrobial Agents and Chemotherapy39 , 476 –83[Abstract]

Received 9 July 1998; returned 11 August 1998; revised 28 August 1998; accepted 24 December 1998