Experimental immunology
The effect of selected cephalosporins on angiogenic activity of human blood mononuclear cells

Introduction
The word antibiotic comes from the Greek anti meaning “against” and bios meaning “life”. Selective toxicity is the essence of antibiotic mode of action. Antibiotic should destroy or inhibit bacterial (or other microorganism) growth not affecting their host organism. Hovewer, it is known that these drugs may disturb biological homeostasis of organism manifesting adverse or side effects [1]. There is a need to investigate this unwanted influence to manage antibiotic therapy in a proper way. Interestingly, suppression of some immunological parameters caused by antibiotic administration can be beneficial in some specific disorders.
Angiogenesis, a new blood vessels formation from existing one, is a necessary process for proper functioning of the organism. Angiogenesis can be characteristically disturbed in many diseases. In malignancy or arthritis angiogenic activity is too high, while in e.g. coronary heart disease it is diminished [2]. So in the first case during bacterial infection it would seem reasonable to use the drug which decreases angiogenesis, while in the second one the drug enhancing angiogenesis is more desirable. So, the doctor should knows patients immunological status to aplicate him a therapy which compensates disturbed immune defence.
Cephalosporins belong to the most frequently used class of antibiotics. They are highly active against wide spectrum of bacteria, reaches effective concentrations in blood and respiratory secretions, giving infrequent adverse effect (mainly hypersensitivity reaction 1-5% and nonspecific gastrointestinal reaction 5-10%) [3].
Te aim of our study was to evaluate the influence of some cephalosporin antibiotics on neovascular reaction induced in mice by intradermal grafting of healthy volunteers mononuclear cells.


Materials and methods
All experiments were performed with mononuclear cells (MNC) obtained from peripheral blood of 12 healthy volunteers which signed Informed Consent.
The following antibiotics were tested belonging to cephalosporin group:
• Sefril (Cefradine, Polfa, Poland)
• Biotrakson (Ceftriaxone, Glaxo, USA)
• Pseudomonil (Cefsulodin, Ciba, Denmark)
• Biocefal (Cefuroxime Glaxo, USA)
• Biofort (Ceftazidime, Glaxo, USA)
• Cefobid (Cefoperazone Pfizer, USA)
Daily doses of antibiotics (3, 15, 75 mg/kg mice body mass) corresponded with the doses used in human therapy.


Isolation of MNC
Mononuclear leukocytes (MNC) were isolated from blood by centrifugation on Ficoll/Uropoline gradient, according to Boyum [4]. The viability of isolated cells was determined by trypan blue exlusion and was always higher than 98%.


Monocyte elimination
MNC suspension was mixed with carbonyl iron (15 mg/107 cells) and incubated for 1 h, at 37°C in plastic bottles. Non adherent cells were poured into the tubes, and phagocytic and lymphoid cells were separated using a magnet. The lymphoid cells (0-2% peroxidase possitive, less then 3% BMA 0310 positive) were washed twice and resuspended in PBS – FCS or Parker medium.


CD8 positive cells elimination
Monocyte – depleted cell suspensions (2 × 107 cell/ml) were incubated for 30 min at 4°C with 5 µg/ml monoclonal antibody (BMA 081). Then the cells were washed twice and resuspended in PBS medium supplemented with 2% inactivated FCS. Cell suspensions were placed on plastic Petri dishes precoated with affinity purified rabbit anti-mouse IgG for 30 min at room temperature and then 30 min in 37°C, and washed in PBS. Cell suspensions enriched with CD4+ cells contained less then 1% CD8+ cells, as estimated by APAAP method.


Angiogenesis test
Angiogenesis test was performed according to Sidky and Auerbach [5] with some modifications [6]. All experiments were performed on inbred 6-8 week old Balb/c mice. Various fractions of human MNC were injected intradermally (5 × 105 per inoculum) into anesthetized mice .Afterwards, antibiotics were administered subcutaneously into the mice (3 mice in one group) for 3 consequitive days at the dose of 3, 15 and 75 mg/kg of the body mass. Mice from the control group were injected with PBS. Each experiment was repeated at least 2 times. After 72 h mice were sacrificed and newly formed blood vessels were counted in dissection microscope (magnification 6 ×) on the inner skin surface.
Experiments were approved by Local Ethical Com­mittee.


Statistical analysis
Angiogenic activity was calculated as a number of newly formed blood vessels and presented as an index (I) counted from the formula: I = a/b, (a: newly formed blood vessels in tested group; b: mean number of newly formed blood vessels in the control group)
All data are shown as a mean ±SEM. The differences between the groups were evaluated by two-way ANOVA and Bonferroni post-test to compare replicate means by rows. The differences were considered significant at p-value < 0.05. Evaluation of the results of experiments with cell fractions was done by one-vay ANOVA and Tukey’s post-test.


Results
Results were shown in Tables 1, 2, Figures 1, 2.
Cephalosporins exerted different effects on angiogenic activity of MNC, depending on type of antibiotic and its dose (Figure 1, Table 1). Inhibitory action was noted for 3 antibiotics from this group. The strongest effect was obtained for cefradine which diminished angiogenic response in each used dose. The smallest number of new blood vessels was observed for 15 mg/kg (p < 0.001). Cefsulodin slighty (p < 0.05) decreased angiogenesis at doses 15 and 75 mg/kg, while 3 mg/kg did not differ from the control group. Lowering (p < 0.05) of angiogenesis level was observed also for the highest dose (75 mg/kg) of ceftriaxone. There was no effect of cefoperazone on angiogenesis test. Cefuroxime and ceftazidime stimulated angiogenic response. In the case of cefuroxime this effect was observed for doses 3 and 75 mg/kg (p < 0.01). Significant (p < 0.001) stimulatory effect was produced by 15 mg /kg of ceftazidim dose, while the doses 3 and 75 mg/kg didn’t change the level of angiogenic response. Studies concerning mechanism of cefradine inhibitory action were performed using the most inhibitory dose of this drug (15 mg/kg) (Table 2, Figure 2). Depletion of monocytes from MNC suspension caused slight increase of new blood vessels number, while depletion of monocytes and CD8+ cells caused further growth of angiogenesis level. The differences between groups were statistically significant (p < 0.001).



Discussion
Cephalosporins belong to the group of antibiotics with heterogeneous influence on immune function. Cephalo­sporins of the first (like cefradine) and second generation (cefuroxime) were considered by many authors inhibitory or neutral for immune response, while cephalosporins of third generation were seen as rather stimulating. In our studies cefradine inhibited angiogenic activity of MNC in all used doses. Studies concerning cefradine immunological effect presented by Chaperon et al. [7] and Ogawa et al. [8] are in line with our results. They revealed that cefradine administration inhibited lymphocyte response to PHA and Con A and decreased production of anti-SRBC antibodies by mouse splenocytes. The inhibition produced by cefradine, in the context of our present results, might have arisen from the stimulation of CD8+cells and monocytes which, in our experiments, synergically diminished angiogenic response of MNC. Similar effects were obtained by Eismary group [9]. They revealed that suppression activity of CD8+cells was associated with the presence of monocytes and CD4+cells. Cefsulodin was shown [10] to decrease humoral response in mouse model. Inhibitory effect of this drug used at therapeutic doses (15 and 75 mg/kg) was obtained also in our work.
Ceftriaxone in our experiments diminished angiogenic response at dose 5 times higher (75 mg/kg) then therapeutic. Subminimal concentration of ceftriaxone, not studied in our paper, according to Raponi et al. [11] increased polymorphonuclear leucocytes (PMN) phagocytic activity of Klebsiella oxytoza and Staphylococcus aureus.
Cefuroxime exerted stimulatory effect on angiogenic response at doses 3 and 75 mg/kg, as demonstrated. Chemotaxis of leukocytes and their spontaneous activation was enhanced by administration of cefuroxime. This drug did not influenced phagocytosis and fungicidal activity. Increased production of IgM antibodies but not IgG by BDF and Balb/c nu/nu mice as well as no influence on PWM induced leucocytes migration were presented for cefoperazone [12]. In our present work this antibiotic was the only drug that didn’t influenced neo-vascular reaction.
Our previous studies [13] revealed stimulatory effect of cefoperazone on angiogenic activity of bronchoalveolar lavage cells obtained from sarcoidosis patients. We have also shown that angiogenic activity of lung cancer cells was diminished after cefoperazone and cefuroxime administration. The same drugs inhibited angiogenesis induced by ovarian cancer cells [14]. Such observations suggested that these cephalosporins can regulate production of angiogenic growth factors. Administration of these drugs could be beneficial during bacterial infections in cancer patients.
There is a lot of studies of ceftazidime effects on immune defences. In our experiments ceftazidime enhanced angiogenesis only at 15 mg/kg – the dose 2 times lower than therapeutic. Stimulation of immune parameters was observed for subminimal concentration of this drug. Cuffini et al. [15] demonstrated enhanced phagocytic and bactericidal activity of human and mouse macrophages after administration of subminimal doses of this antibiotic. Elevation of humoral response and bactericidal activity of macrophages was obtained after administration of 2,5-3 mg/kg ceftazidime in Balb/c mice. Other in vivo and ex vivo ceftazidime studies indicated stimulation of phagocytosis, production of IL-1 and IFN-γ in immunologically impaired patients and laboratory animals [16]. Single dose of ceftazidime enhanced blood concentration of some pro-inflammatory cytokines (IL-6 and TNF-α) in bloodstream of rats [17]. However supratherapeutic concentrations of ceftazidime inhibited production of TNF-α by human PBMC stimulated with heat-killed Stenotrophomonas maltophilia [18].

Conclusions
1. Cephalosporins exerted heterogeneous effect on angiogenic MNC response depending on the type of antibiotic and their dose.
2. The mechanism of inhibition probably rely on activation of suppressor/cytotoxic function of CD8 positive cells with monocytes cooperation.
3. Cefradine and cefsulodin are suggested to exert a beneficial effect in therapy of patients with high level of pathological angiogenesis, while cefuroxime and ceftazidime are expected to enhance immunological and angiogenic response of angio- and immuno-compromised patients.


Acknowledgements
This work was partly performed in Institute of Tuberculosis and Lung Diseases, Płocka 26, Warsaw, Poland.


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