Departments of 1 Biomedical Sciences and 2 Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Trieste I-34127, Italy
Received 12 February 2002; returned 5 May 2002; revised 28 May 2002; accepted 10 June 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One potentially useful systemic application for AMPs concerns fungal infections, due to the increased incidence of mycoses particularly in immuno-depressed individuals.19 As ethical reasons restrict animal employment in the development of in vivo models of efficacy or toxicity, the preliminary use of in vitro methods based on the host cells likely to be encountered by the drug candidate, such as, for example, circulating leucocytes in the case of intravenous (iv) use, is desirable. We have studied the cytotoxic activity of three artificial -helical AMPs on mouse primary cultures of resting or activated lymphocytes. These compounds were designed as part of a detailed structureactivity relationship study of the effect of physicochemical characteristics on the in vitro antimicrobial activity, specificity and selectivity of this type of small linear AMP.3,20 Peptide- and concentration-dependent membrane damage was quantitatively determined using flow cytometric methods and confirmed by electron microscopy. These data indicated the most appropriate peptide and dose likely to provide active protection from an infecting pathogen while minimizing the toxic effect on circulating host cells. The effectiveness of this peptide in vivo was assessed in a limited mouse candidiasis protection study.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The peptides P19(8), D-P19(9/B) and L-P19(9/B) were designed using a sequence template obtained by analysing numerous naturally occurring -helical peptides, as described previously.20 Solid-phase syntheses were carried out on an automated synthesizer (Pioneer PerSeptive Biosystems) using PEG-PS resins (0.170.22 mEq/g) and a six-fold excess of 1:1 Fmoc-amino acid/TBTU [where TBTU corresponds to O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate] with di-isopropylethyl amine as base, as described. Peptides were purified by preparative reversed-phase (RP) HPLC [Waters (Milford, MA, USA) RadialPak, 5 µm, 300 Å, 10 x 150300 mm], and their correct structure and purity were determined by mass determination with an API I ion-spray instrument (Applied Biosystems SCIEX). Peptide concentrations were quantified using tyrosine absorption (
280 = 1290 M1·cm1). Circular dichroism (CD) spectra were obtained on a Jasco J-700 dichrometer using 2 mm path-length cuvettes and peptide concentrations of 4 x 105 M, in 5 mM phosphate buffer pH 7, in the presence of 50% trifluoroethanol or 10 mM SDS, and the percentage helicity was determined as ([
]meas [
]rc)/([
]
[
]rc), where [
]meas is the measured ellipticity at 222 nm, [
]rc is the ellipticity for unstructured peptide in the absence of additives (generally close to zero) and [
]
is the ellipticity of a fully structured helix of length n, calculated using the relation [
]
= 39 000 (14/n).21 The presence of aminoisobutyric acid (Aib), which is achiral, was taken into account in these calculations.22 Stock solutions of peptide were obtained by resuspending lyophilized peptide in phosphate-buffered saline (PBS) and diluting as appropriate in PBS for all experiments.
Haemolytic activity
Lysis of erythrocyte membranes was determined by monitoring the release of haemoglobin at 415 nm from 0.5% human erythrocyte suspensions (0.2 mL) in PBS, in relation to complete (100%) haemolysis as determined by addition of 0.2% Triton X-100.
Preparation of lymphocyte single-cell suspensions
Lymphocytes were obtained from spleens of healthy mice. The organ was placed on a sterile Petri dish with 5 mL of RPMI5% fetal calf serum (FCS) and pressed with the barrel of a plastic syringe until complete tissue disaggregation was achieved. The cell suspension was filtered through a sterile gauze to remove debris, and centrifuged at 400g for 5 min at 4°C. Red blood cells were removed by hypotonic shock, and following a further centrifugation, lymphocytes were re-suspended with PBS, counted by the Trypan Blue exclusion test and diluted to the appropriate concentration with PBS or complete medium [CM: RPMI 1640 (Sigma Chemical Co.), 10% fetal bovine serum (FBS; Euroclone), 1% penicillin/streptomycin 100x (Euroclone)] for the in vitro tests.
T-cell lymphoblasts were obtained from spleen cells, prepared as described above and dispersed at 1 x 106/mL in 10 mL of CM in upright 50 mL culture flasks. After addition of 2.5 µg/mL concanavalin A (ConA), flasks were incubated for 72 h, followed by two washes with PBS, and activated cells were recovered and counted by the Trypan Blue exclusion test.23 Lymphoblasts were identified by cell cycle, phenotypic analysis and mitochondrial respiration (JC probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; Molecular Probes, Eugene, OR, USA).
Flow cytometric analysis
Suspensions of 106 lymphocytes in 1 mL of PBS were exposed to the desired peptide (4160 µM) for 30 or 60 min at 37°C. Bulk phase peptide was then removed by centrifugation at 400g for 5 min at 4°C followed by double washing with PBS. Samples were prepared at least in triplicate.
Propidium iodide uptake
At the end of the incubation time washed cells were diluted with PBS and added to 10 µL of propidium iodide (PI) solution (0.5 mg/mL in PBS) immediately prior to flow cytometric analysis. Fluorescence data from damaged cells showing permeability to PI were acquired in a monoparametric histogram (emission wavelength = 612 nm).
Dual staining FITC/PI
Cells (5 x 105) were fixed in 70% EtOH for at least 4 h, then washed twice and allowed to balance in PBS for 2 h. Pellets were resuspended in a 1 mL PBS solution containing 10 µg of PI, 0.05 µg of fluorescein isothiocyanate (FITC) and 4 µg/mL RNase (all from Sigma Chemical Co.). Cells were stained overnight before flow cytometric analysis.
Monoclonal antibody staining
Samples of lymphocyte suspensions obtained from 24 h in vitro cultures were washed, resuspended with PBSNaN3bovine serum albumin (BSA) and stained with fluorescent monoclonal antibodies (MoAb): CD3-FITC (1 µg/106 cells) and CD19-PE (phycoerythrine) (0.5 µg/106 cells) (Southern Biotecnology Associates Inc., Birmingham, AL, USA) for 30 min, and unbound antibodies removed by two washes. Samples treated with an irrelevant isotype matched and FITC/PE MoAb, run in parallel, were used to set the gates in the monoparametric histogram to include <2% aspecific fluorescence events. All flow cytometric analyses were performed with an XL instrument (Coulter Inst., Miami, FL, USA). At least 10 000 events were acquired for each sample. Histograms were analysed with the WinMDI software (Dr J. Trotter, Scripps Research Institute, La Jolla, CA, USA).
Scanning electron micrographs
Lymphocyte suspensions, after challenge with the test peptides, as described above, were re-suspended in PBS and diluted to 20 x 106 cells/mL. A drop of each cell suspension was layered on to slides coated previously with poly-L-lysine solution (0.1 mg/mL, Sigma), and allowed to adhere for 2 h. Cells were then fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer overnight. All procedures were performed at 4°C. Samples were dehydrated in graded ethanol, vacuum dried and mounted on to aluminium scanning electron micrograph (SEM) mounts. After sputter coating with gold, they were submitted for analysis with a Leica Stereoscan 430i instrument.
Animal studies
Animal studies were carried out according to the DHHS Guide for the care and use of laboratory animals.24 Evaluation of the in vivo toxicity was performed by a single iv injection of the test peptides at doses of 12.5, 25 and 50 mg/kg. Systemic candidiasis was induced in healthy BALB/c female mice (Animal House, University of Trieste) by injection into a tail vein of 5 x 105 Candida albicans (H12 strain, ATCC56879) per mouse. This procedure leads to a lethal infection within 10 days in the control group.25 Groups of 810 animals were treated with the test peptide at each dose, or with PBS alone (control). For the lower dose (5 mg/kg) and control the experiment was repeated twice. Peptide was administered as a single dose, and was injected intravenously 6 h after the induction of systemic candidiasis. Animal survival was recorded on a daily basis.
Statistical analysis
Cytometric data were submitted to computer-assisted analysis (Instat II; Graphpad Software, version 2.05) using the Student/NewmannKeuls test for analysis of variance (ANOVA), whereas animal survival data were subjected to the non-parametric KaplanMeier test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
In order to distinguish the cytotoxic effects on B- and T-lymphocytes (which we estimate to represent jointly 80% of the splenocyte suspension23), fluorescently labelled anti-CD19 and anti-CD3 monoclonal antibodies were used to determine the respective percentages in recovered viable cells, 24 h after treatment with P19(8) for 3060 min (Table 3). These cells thus resisted permeabilization in the first hour of exposure to peptide. At concentrations of up to 40 µM, this peptide did not affect the relative proportion of undamaged B- and T-lymphocytes (roughly 1:1), as compared with an untreated control, whereas at 160 µM, the proportion of B-lymphocytes was significantly reduced (
30% of the lymphocyte population). Undamaged cells did not appear to show a different distribution among cell cycle phases from the untreated control (based on the fluorescence characteristic of cells, as assessed by PI DNA staining after ethanol permeabilization, Table 3), independently of the challenge dose or time of exposure. Furthermore, 24 h after in vitro treatment with P19(8), undamaged cells had a similar proportion of viable cells to sub-G1 cells (see the % G0/G1 cells, Table 3), and a similar protein content in viable cells to that in the control (as assessed by FITC fluorescence, Table 3). That exposure to the peptide did not induce apoptosis was also suggested by the fact that the respiratory activity of mitochondria in surviving lymphocytes treated with 16 µM P19(8) for 60 min was not significantly different from untreated cells as determined using the JC-1 probe (data not shown).
|
In order to relate the in vitro cytotoxicity data with in vivo effects, a limited number of toxicity experiments were performed on a BALB/c mouse model, administering the least and most cytotoxic peptides, P19(8) and L-P19(9/B), at doses varying from 12.5 to 50 mg/kg, corresponding to estimated blood concentrations of 80300 µM immediately after injection (respectively about half and twice the maximal dose used in flow cytometry experiments). These concentrations are purely indicative, as the peptide blood level is likely to be rapidly reduced by binding to cellular and plasma components. Only the higher dose was lethal in both cases, while all animals survived at 25 mg/kg. At the lower doses both peptides induced visible signs of toxicity (difficulty in breathing and movement, ruffled fur), but the time of onset of suffering was considerably longer and that of recovery shorter for P19(8) than for L-P19(9/B), indicating a lower toxicity for P19(8) also in vivo.
On this basis, and in consideration of the in vitro effects, P19(8) was considered to be the most suitable candidate for use in a mouse systemic candidiasis protection model. The survival analysis of mice infected with C. albicans was carried out at a dose of 5 mg/kg peptide administered iv 6 h after the induction of infection. This dose is 10 times lower than the lethal dose, and in fact no visible signs of toxicity were observed. It corresponds to an estimated blood concentration of 30 µM immediately after administration, which is two to four times the in vitro MIC, while corresponding to a relatively contained cytotoxic activity on circulating cells as assessed by the in vitro studies. Again, this concentration is purely indicative for the reasons discussed above. The survival analysis is shown in Figure 4, and indicates a modest but statistically significant shift of the curve towards a longer survival. Interestingly, higher doses of the peptide (10 and 20 mg/kg) did not improve survival, but conversely decreased it with respect to untreated mice at the higher dose.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have determined how these structural variations affect the cytotoxicity of the peptides on other types of blood cells, in a murine model, with a view to evaluating their potential for systemic applications, such as, for example, in the treatment of systemic candidiasis. Flow cytometry is a rapid and powerful method for the in vitro evaluation of cytotoxicity, as analysis of PI fluorescence allows for an accurate quantification of damage to circulating cells by cytolytic AMPs, and various molecular tools are available to distinguish this effect in different cell types. PI uptake shows that permeabilization of the cytoplasmic membrane occurs with all three peptides tested (Tables 2 and 4), and the variation of morphological parameters (reduction of both forward and side scattering; Figure 2) would seem to indicate that this damage results in necrosis. Membrane damage is also supported by the visible morphological alterations to the cellular surface observed by SEM, such as bleb formation and eventually cellular collapse (Figure 3). There is, however, both a quantitative and a qualitative difference in damage caused by the three peptides under consideration. (i) The proportion of damaged cells is significantly lower for P19(8) than the other two peptides; (ii) damage to resting and activated lymphocytes is comparable after exposure to P19(8) but greater for activated cells after exposure to the other two peptides (Tables 2 and 4), at equivalent concentrations; (iii) essentially no variation for the cytotoxic effect was observed after 30 or 60 min exposure to P19(8), in contrast to the two P19(9/B) enantiomers, where damage was significantly greater at the longer time (Table 4); (iv) the D and L enantiomers of P19(9/B) appear to have an appreciably different effect on host cells (both erythrocytes and lymphocytes). This suggests that the mechanism of eukaryotic membrane permeabilization by the three -helical AMPs may differ somewhat, in a manner that depends principally on the propensity for helical structuring, which is lower in P19(8) than in the P19(9/B) enantiomers, while charge, hydrophobicity and amphipathicity are substantially similar. Furthermore, it also appears to depend on the stereochemistry of the peptide. This feature of
-helical AMPs has not to our knowledge been reported previously, and could be useful, as unlike modulation of helix-forming propensity, changing the helical sense has no effect on antimicrobial potency.3
The fact that P19(8) was less cytotoxic in all in vitro experiments, and did not result in increased damage to cells at the longer exposure time, even at the highest concentration used, suggested that it might be the best candidate for in vivo studies, despite its somewhat lower antimicrobial potency. Notably, even at the highest concentration used (160 µM), a significant proportion (up to 20%) of exposed cells survived challenge by this peptide, as observed both by flow cytometric analysis and SEM. This could mean that a proportion of the leucocyte population may have membranes less susceptible to damage by the peptide, related either to the cell type or the phase of the cell cycle. In this respect, T-lymphocytes appear to be more resistant to exposure to high concentrations of P19(8) than B-lymphocytes, whereas there appears to be little difference in its effect on resting or ConA-activated T-lymphocytes.
Conversely, ConA-activated lymphocytes were more susceptible than resting cells to the more cytotoxic peptide D-19(9/B), which is reminiscent of the different cytotoxicity displayed by the endogenously produced human -helical peptide LL-37 on resting blood cells and the proliferating MOLT T-lymphocyte cell line.17 Another example of differential toxicity has been reported for the endogenously produced
-helical bovine myeloid antimicrobial peptides (BMAP) with respect to T-lymphocytes from healthy individuals and those from patients with different types of leukaemia, which were considerably more susceptible.31 Furthermore, this study indicated that at low concentrations (6 µM) the BMAP peptides induce apoptosis in activated lymphocytes. In our study, after exposure to P19(8) no apparent effects were observed on surviving washed cells relative to unexposed cells, after 24 h (as judged by DNA and protein content), further indicating that under our conditions inactivation is probably by an early necrotic effect rather than a subsequent apoptotic induction.
On the basis of the in vitro data, a limited in vivo study was carried out to test the toxicity and antimycotic efficacy of P19(8) in a mouse model. The peptide appeared to be relatively well tolerated iv up to a single dose of 12.5 mg/kg. We judged that for a 5 mg/kg peptide administration, the maximal serum concentration immediately after injection (30 µM) might be sufficiently high for measurable antimycotic activity, without resulting in marked depletion of host immune cells, even taking into account the likely rapid reduction in bioavailability of the peptide due to binding to cells and plasma components. Treatment 6 h after an infection with 5 x 105 C. albicans cells did result in a modest but statistically significant increase in survival, indicating that the peptide did in fact have a protective effect, reducing the infecting charge of the yeast cells but not sufficiently to save treated animals. On the other hand, at higher non-lethal doses (10 and 20 mg/kg), survival was, respectively, unaffected and actually decreased with respect to untreated animals. This could be an indication that damage to leucocytes by the peptide at these higher doses may counteract a greater antimicrobial effect by decreasing the animals defence capacities.
An in vivo study on a mouse aspergillosis model has been reported for the potent tryptophan-rich antimicrobial peptide indolicidin, which appears to be considerably more toxic than P19(8). It was ineffective at prolonging the survival time at a dose equivalent to that at which P19(8) shows some effect.16 Liposomal entrapment of this peptide, however, considerably decreased its cytotoxicity and allowed administration of doses up to 40 mg/kg with a marked increase in efficacy that resulted in 30% long-term survival. This may also be a useful way of mitigating the cytotoxicity of -helical AMPs.
From our study, we conclude that the in vitro cytotoxicity of the linear -helical AMPs tested towards blood cells depends principally on structural characteristics. Modulating the structure to reduce cytotoxicity has allowed for a marginal window of success with respect to the in vivo use of the less toxic peptide in the treatment of systemic candidiasis in a mouse model. While this window might be extended by further rationally modulating structural characteristics of the peptides so as to boost the antimycotic activity without increasing cytotoxicity, any serious consideration of their systemic use would also require improvement of pharmacological properties by other means, such as, for example, appropriate formulation as described for indolicidin above. If this is achieved, a potentially useful application of AMPs at low concentrations could be in conjunction with clinically used antibiotics, especially against pathogens that have become resistant to them. Some studies have in fact shown that synergy of action can exist between clinically used antibiotics/antimycotics and AMPs.31
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Hancock, R. E. & Scott, M. G. (2000). The role of antimicrobial peptides in animal defenses. Proceedings of the National Academy of Sciences, USA 97, 885661.
3 . Tossi, A., Sandri, L. & Giangaspero, A. (2000). Amphipathic, alpha-helical antimicrobial peptides. Biopolymers 55, 430.[ISI][Medline]
4 . Tossi, A. & Sandri, L. (2002). Molecular diversity in gene- encoded, cationic antimicrobial polypeptides. Current Pharmaceutical Design 8, 74361.[ISI][Medline]
5 . Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature 415, 38995.[ISI][Medline]
6 . Matsuzaki, K. (1999). Why and how are peptidelipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochimica et Biophysica Acta 1462, 110.[ISI][Medline]
7 . Shai, Y. (1999). Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochimica et Biophysica Acta 1462, 5570.[ISI][Medline]
8
.
De, Y., Chen, Q., Schmidt, A. P., Anderson, G. M., Wang, J. M., Wooters, J. et al. (2000). LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. Journal of Experimental Medicine 192, 106974.
9 . Yang, D., Chertov, O. & Oppenheim, J. J. (2001). The role of mammalian antimicrobial peptides and proteins in awakening of innate host defenses and adaptive immunity. Cellular and Molecular Life Sciences 58, 97889.[ISI][Medline]
10 . Nizet, V., Ohtake, T., Lauth, X., Trowbridge, J., Rudisill, J., Dorschner, R. A. et al. (2001). Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414, 4547.[ISI][Medline]
11 . Chen, J., Falla, T. J., Liu, H., Hurst, M. A., Fujii, C. A., Mosca, D. A. et al. (2000). Development of protegrins for the treatment and prevention of oral mucositis, stucture-activity relationships of synthetic protegrin analogues. Biopolymers 55, 8898.[ISI][Medline]
12 . Hancock, R. E. W. (2000). Cationic antimicrobial peptides, towards clinical applications. Expert Opinion on Investigational Drugs 9, 17239.[ISI][Medline]
13 . Lamb, H. M. & Wiseman, L. R. (1998). Pexiganan acetate. Drugs 56, 104752.[ISI][Medline]
14 . Paquette, D. W., Waters, G. S. & Stefanidou, V. L. (1997). Inhibition of experimental gingivitis in beagle dogs with topical salivary histatins. Journal of Clinical Periodontology 24, 21622.[ISI][Medline]
15
.
Sorensen, O., Bratt, T., Johnsen, A. H., Madsen, M. T. & Borregard, N. (1999). The human antibacterial cathelicidin, hCAP-18, is bound to lipoproteins in plasma. Journal of Biological Chemistry 274, 2244551.
16 . Ahmad, I., Perkins, W. R., Lupan, D. M., Selsted, M. E. & Janoff, S. A. (1995). Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity. Biochimica et Biophysica Acta 1237, 10914.[ISI][Medline]
17
.
Johanson, J., Gudmundsson, G. H., Rottenberg, M. E., Berndt, K. D. & Agerbeth, B. (1998). Conformation dependent antibacterial activity of the naturally occurring human peptide LL-37. Journal of Biological Chemistry 237, 371824.
18 . Skerlavaj, B., Benincasa, M., Risso, A., Zanetti, M. & Gennaro, R. (1999). SMAP-29, a potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Letters 463, 5862.[ISI][Medline]
19 . Fromtling, R. A. (1998). Human mycoses and current antifungal therapy. Drug News Perspectives 11, 18591.[ISI]
20
.
Giangaspero, A., Sandri, L. & Tossi, A. (2001). Amphipathic -helical antimicrobial peptides, a systematic study of the effects of structural and physical properties on biological activity. European Journal of Biochemistry 268, 5589600.
21 . Chen, Y. H., Yang, J. T. & Chau, K. H. (1974). Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry 13, 33509.[ISI][Medline]
22
.
Gobbo, M., Bioni, L., Filira, F., Formaggio, F., Crisma, M., Rocchi, R. et al. (1998). Helix induction potential of N-terminal -methyl,
-amino acids. Letters in Peptide Science 5, 1057.[ISI]
23 . Klaus, G. B. (1987). Lymphocytes. A Practical Approach. IRL Press, Oxford, UK.
24 . Department of Health and Human Services. (1985). Guide for the Care and Use of Laboratory Animals. pp. 2386. DHHS Publications, National Institutes of Health, Bethesda, MD, USA.
25 . Polak, A. (1998). Experimental models in antifungal chemotherapy. Mycoses 41, 130.[ISI][Medline]
26 . Lewis, R. S. & Cahalan, M. D. (1995). Potassium and calcium channels in lymphocytes. Annual Reviews in Immunology 13, 62353.[ISI][Medline]
27 . MacDonald, R. & Nabholz, M. (1986). T-cell activation. Annual Reviews in Cell Biology 2, 23153.
28 . Tossi, A., Tarantino, C. & Romeo, D. (1997). Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity. European Journal of Biochemistry 250, 54958.[Abstract]
29 . Tiozzo, E., Rocco, G., Tossi, A. & Romeo, D. (1998). Wide-spectrum antibiotic activity of synthetic amphipatic peptides. Biochemical and Biophysical Research Communications 249, 2026.[ISI][Medline]
30 . Risso, A., Zanetti, M. & Gennaro, R. (1998). Cytotoxicity and apoptosis mediated by two peptides of innate immunity. Cellular Immunology 189, 10715.[ISI][Medline]
31 . vant Hof, W., Reijnders, I. M., Helmerhorst, E. J., Walgreen-Weterings, E., Simoons-Smit, I. M., Veerman, E. C. et al. (2000). Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity. Antonie Van Leewenhoek 78, 1639.[ISI][Medline]