THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol. 22 (1), 2015
Page: 85-91
Immunomodulatory Effects of Levofloxacin on Patients with Pneumonia in Assiut University Hospitals
1Mohamed S. Badari, 1Sherein G. Elgendy, 1Asmaa S. Mohamed, 2Alaa
T. Hassan
Departments of 1Medical Microbiology Immunology, Faculty of Medicine, and 2Chest Diseases, Assiut University hospitals, Assiut University, Assiut, Egypt
The immunomodulatory effects of antibiotics could influence the degree of systemic and local responses to infection, so investigation of their intrinsic influence on the host’s inflammatory response appears to be essential. Fluoroquinolones are known to exert modulatory activity on immune responses to microbial infection. However the mechanism of this immunmodulation has not been well elucidated. The aim of the work, is to assess the immunomodulatory effects of a levofloxacin, through examining its effect on the concentrations of tumor necrosis factor α (TNF-α) and Interleukin – 10 (IL-10) in serum of pneumonic patients. After following local research ethics committee approval and informed consent. This study included
40 patients with different types of pneumonia, admitted to department of Chest Diseases, Faculty of Medicine, Assiut University Hospitals, Egypt. Also, 10 healthy volunteers served as randomized controls. Both patients and controls received levofloxacin (750 mg once daily for 10 days). Serum levels of TNF-α and IL-10 were measured in patients and control before and after levofloxacin administration (750 mg once daily for 10 days) using human TNF–α and IL-10 ELISA kits respectively. Levofloxacin caused a statistically significant decrease in the mean level of TNF- α in both patients (20.82±1.31 pg/ml) (P 0.009) and control group (17.12 ±0.84 pg/ml) (P 0.004). In contrast, there was statistically significant increase (P< 0.000) in the mean level of IL-10 in patients (61.75 ± 2.85 pg/ml) while statistically significant decrease (P 0.005) in control group (28.57 ± 1.37pg/ml). In conclusion, our study demonstrates that treatment with levofloxacin affects production of TNF-α as a pro-inflammatory cytokine and IL-10 as an anti-inflammatory cytokines which may provide additional benefits in treatment of respiratory tract infections that are independent of its antibacterial properties.
neumonia is a leading cause of death in the world and the sixth most common cause of death in the United States. It is
the number one cause of death from infectious diseases in the United States. In Europe, the overall incidence of community acquired lower respiratory tract infections (LRTIs) was found to be 44 cases per 1,000 populations per year in a single general practice. However, the incidence was two to four times higher in people aged over 60 years (Wei et al., 2009; Woodhead et al., 2005).
There is growing evidence that certain antibiotics exert their beneficial effects not only by killing or inhibiting the growth of bacterial pathogens but also indirectly by their up regulatory effect on immune system. It has
been noted that certain antibiotics (macrolides and fluoroquinolones) have immuno- modulatory properties that improve the long term outcome of patients with inflammatory pulmonary diseases (Tauber & Nau, 2008).
Levofloxacin is one of the newest third generation fluoroquinolones, it is the bacteriologically active L-isomer of ofloxacin (quinolone antibacterial agent). Levofloxacin has a broad spectrum of action, it diffuses through bacterial cell wall and acts by inhibiting and disrupting the function of DNA gyrase (bacterial type II topoisomerases) leading to blockage of bacterial cell growth. So levofloxacin acts as an efficient anti- bacterial agent by hijacking the natural ability
of topoisomerase to create breaks in chromosomal DNA (Najma et al., 2009).
Levofloxacin is a highly appropriate agent for treating respiratory tract infections due to its broad anti-bacterial spectrum of action for all of the most common respiratory tract pathogens, being effective against Gram- negative and Gram-Positive, as well as atypical organisms. Also its excellent pharmacokinetic and pharmacodynamic features which allows it to penetrate extremely well into lung tissue and bronchial secretions. In addition to its ability to penetrate into both phagocytic and epithelial cells which appears to be extremely important in inhibition of intracellular organisms. It achieves high concentrations in respiratory secretion and lung tissue and has a persistent activity in lung tissue “post antibiotic effect” (Carl, 2000).
Fluoroquinolones have immunomodulatory effects that are independent on its antibacterial properties. The molecular mechanisms causing immunomodulatory effects are still under investigations. However activation of p38 mitogen-activated protein kinase (MAPK) pathway which is considered one of the major signal transduction pathways involved in inflammatory responses was proposed as the main effect of fluoroquinolones (Tauber and Nau, 2008).
In the in vitro studies, fluoroquinolones exert their modulating effects only when used together with a co-stimulant. These studies generated heterogeneous data because of inhomogeneous effects triggered by different types of co-stimulants and differing responses of various cell lines on the stimuli. Studies in experimental animals showed significant clinical effects of flouroquinolones by attenuating cytokine responses in vivo (Dalhoff, 2005).
The first study on the immunomodulatory activity of fluoroquinolones were independent on drug concentration and the analytical
methods to quantitate cytokines were less sensitive, so the modulations of cytokines synthesis due to exposure to fluoroquinolones remained undetected. The older quinolone like nalidixic acid, enoxacin, fleroxacin, norfloxacin, and ofloxacin super induce cytokine synthesis at high concentration using human peripheral blood lymphocytes stimulated with phytohaemaglutinin, however, exposure to other stimulants led to inhibition of cytokine synthesis (Bailly et al., 1990)
Ciprofloxacin inhibited I1-1α and I1-1 ȕ synthesis in lipopolysaccharide stimulated human peripheral blood lymphocytes but it augmented I1-1 synthesis in lipopolysaccharide stimulated Mono-Mac6 cells (Stunkel et al., 1991). Trovafloxacin significantly inhibited the secretion of I1-1α, I1- ȕ and GM-CSF and TNF-α by monocytes stimulated by LPS (Khan et al., 1998).
Obviously, considerable study variations do exist due to differences in drug concentrations and stimulants used. In addition, the immunomodulatory effect of levofloxacin is not well studied and only few reports were done to evaluate immunomodulatory effect of levofloxacin on pro-inflammatory cytokine production however the experiments were carried out only in vitro. In addition, no data is available to describe the effects of levofloxacin on the anti-inflammatory cytokines. Therefore, this study aimed to evaluate the immunomodulatory effects of levofloxacin through examining its effect on the concentrations of TNF-α and IL-10 in serum of pneumonic patients and control group before and 10 days after its administration.
Material and Methods
Ethics Statement: Written informed consent was obtained from all patients and controls at the time of enrollment for their participation in the study. The study protocol was approved by the local Ethics Committee of the Faculty of Medicine, Assiut University.
The study was conducted during the period from April 2011 to June 2013 as cooperation between Chest and Medical Microbiology Immunology departments, Faculty of Medicine, Assiut University. Blood samples were withdrawn from 40 patients (28 males 12 Females) including 15 patients with community acquired pneumonia (CAP), 12 patients with hospital- acquired pneumonia (HAP) and 13 patients with Ventilator-associated pneumonia (VAP). Patients with HAP and VAP were admitted due to reasons other than infection like pulmonary embolism, pneumothorax and bronchial asthma. In addition, 10 healthy volunteers served as randomized controls. Both patients and controls received levofloxacin (750 mg once daily for 10 days). Blood samples were taken before and 10 days after levofloxacin administration (750 mg once daily). Inclusion criteria: 1- Patients with CAP provided by the BTS criteria for CAP (Wei et al., 2009) which is as follows: Symptoms of an acute lower respiratory tract illness (cough and at least one other lower respiratory tract symptom). New focal chest signs on examination. No other explanation for the illness, which is treated as CAP with antibiotics. Symptoms and signs consistent with an acute lower respiratory tract infection associated with new radiographic shadowing for which there is no other explanation (e.g. not pulmonary edema or infarction).
Patients with HAP and VAP were diagnosed according to The American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) guidelines 2005 that distinguish the following types of pneumonia: Hospital-acquired pneumonia (HAP) is pneumonia that occurs 48 hours or more after admission and did not appear to be incubating at the time of admission. Ventilator-associated pneumonia (VAP) is a type of HAP that develops more than 48 to 72 hours after endotracheal intubation.
Exclusion criteria: Patients with previous antibiotic history in 15 days. Patients with diagnosed malignancies, collagen diseases, DM, hepatitis infection.
All patients were subjected to complete clinical assessment. Routine investigations were performed including; complete blood pictures, erythrocyte sedimentation rates and bacteriological analysis of sputum. Blood samples were obtained under aseptic condition in sterile tubes without any anti-coagulant; each tube was labeled with the patient name, sex, age and the date of collection. Samples were spin down at 2000 r.p.m for 10 minutes; the serum was stored at - 20ºC. Cytokine assay was performed by measuring TNF-α and IL-10 in serum samples using human TNF- α and IL-10 ELISA kit, KOMA BIOTECH INC
(K0331123 and K0331131), respectively. All tests were done according to the manufacturer’s instructions:(a) 200 ml of washing solution were added to each well and washed 3 times. (b) 100 ml of standard (recombinant human TNF-α and IL-10) or samples were added to each well and incubated at room temperature for 2 hours. (c)The wells were aspirated and the plate washed 4 times. (d) 100 ml of the diluted detection antibody (0.5 mg/ml) were added per well, and incubated at room temperate for 2 hours. (e) 100 ml of the diluted color development Enzyme (1: 20 dilute) were added per well, and incubated 30 minutes at room temperate. (f) The plate washed 4 times and 100 ml of color development solution were added and incubated for (8 - 18 minutes). (g) Stop solution was added and the micro plate reader wavelength was set at 450 nm and the absorbance (OD) of each well was measured.
(h) absorbance values of the strandard recombinant human TNF-α and IL-10 samples (supplied with the kit) were used to construct a standard curve from which the concentrations of the cytokines in the tested samples were calculated.
Statistical Analysis
All data were analyzed using the computerized statistical analysis (Statistical package for social science “SPSS version 16”). Concentrations of TNF-α and IL-10 were expressed as mean ± standard error of the mean (SEM). Differences in mean values of TNF-α and IL-10 concentrations before and after levofloxacin administration were calculated using Wilcoxon Signed Ranks Test. Mann–Whitney Test was used for comparison between mean values of patients and control. P-value is considered significant when less than 0.05.
Results
Forty patients with pneumonia including 15 patients with CAP, 12 patients with HAP and 13 patients with VAP were included in this study. Classical Bacteriological examinations showed that 42.5% of the samples showed Gram negative bacteria (Acinetobacter 5.0%, Klebsiella 25%, Pseudomonas 12.5%), while Gram positive bacteria were detected in 57.5% of the samples (MRSA 25.0%,
Pneumococci 32.5%).
This study showed that levofloxacin caused a statistically significant decrease in the mean
level of TNF- α in both patients (P 0.009) and control (P 0.04) as shown in table (1). The mean value of TNF-α in the patients before levofloxacin administration was 36.43
± 4.18 pg/ml while it was 20.82±1.31 pg/ml
after levofloxacin administration. The mean value of TNF-α in control group before levofloxacin administration was 25.21 ± 1.96 while it was 17.12 ± 0.84 pg/ml after levofloxacin administration.
Table 1. Serum TNF-a (pg/ml) in patients with pneumonia and controls.
TNF-a (pg/ml) / Patients / Control / *P1-valueBefore levofloxacin administration / Mean ± SE Median / 36.43 ± 4.18
23.3 / 25.21 ± 1.96
27.8 / NS
Range / 8.2 ± 98.0 / 16.7 ± 33.5
Mean ± SE / 20.82 ± 1.31 / 17.12 ± 0.84
After levofloxacin administration (750mg once daily for 10 days)
Median 18.5 17.9 NS
Range 11.5 ± 46.2 10.7 ± 20.0
*P2-value 0.004 0.009
1: Mann-Whitney Test.
2: Wilcoxon Signed Ranks Test.
* P > 0.05 is not significant (NS)
Regarding IL-10, levofloxacin caused a statistically significant increase in the mean level of IL-10 in patients and a statistically significant decrease in control group as shown in table (2). The mean value of IL-10 in patient before levofloxacin administration was
24.54 ± 2.83 pg/ml, while it was 61.75 ± 2.85 pg/ml after levofloxacin administration. The mean value of IL-10 in control group before levofloxacin administration was 51.48 ± 1.76 pg/ml, while after levofloxacin administration it was 28.57 ± 1.37pg/ml.
Table 2. Serum IL-10 (pg/ml) in patients with pneumonia and controls.
IL-10 (pg/ml) / Patients / Control / *P1-valueMean ± SE / 42.54 ± 2.83 / 51.48 ± 1.76
Before levofloxacin administration / Median / 37.5 / 52.2 / NS
Range / 16.4 ± 79.2 / 40.2 ± 60.5
After levofloxacin administration (750mg once daily for 10 days) / Mean ± SE Median / 61.75 ± 2.85
61.3 / 28.57 ± 1.37
28.6 / 0.000
Range / 20.0 ± 100.1 / 20.0 ± 33.8
*P2-value / 0.000 / 0.005
1: Mann-Whitney Test.
2: Wilcoxon Signed Ranks Test.
* P 0.05 is not significant (NS).
Discussion
Several classes of antibiotics, including macrolides and quinolones exert modulatory effects on cytokine release by inflammatory cells (Parnham Michael, 2005).It is important to define the immunomodulatory
effects of these antibiotics which are commonly used in the therapy of respiratory tract infections, these effects seem to be related to the bacterial killing, as well as, the resolution of local inflammation. This may account for the therapeutic benefit of