Analysis of the cytotoxicity of synthetic antimicrobial peptides on mouse leucocytes: implications for systemic use

Sabrina Pacor1,*, Anna Giangaspero2, Marina Bacac1, Gianni Sava1 and Alessandro Tossi2

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have analysed the toxicity of highly cationic, artificial {alpha}-helical antimicrobial peptides on blood cells to assess their suitability for systemic application. Flow cytometric methods, based on the uptake of propidium iodide, were used to obtain a rapid and quantitative estimate of membrane damage to resting and concanavalin A-activated mouse lymphocytes, which was further confirmed by morphological changes as observed by scanning electron microscopy. Membrane permeabilization appeared to correlate with structural characteristics, so that the peptide L-19(9/B), which contains helix-stabilizing aminoisobutyric acid (Aib) residues and is a potent antimicrobial, was also the most lytic towards both mouse lymphocytes and human erythrocytes. Reducing the propensity for helix formation in P19(8) resulted in a marked reduction in in vitro cytotoxicity. Changing the helical sense in D-P19(9/B) also resulted in a significant decrease in cytolytic activity towards both erythrocytes and leucocytes. A limited assessment in BALB/c mice confirmed a lower in vivo toxicity of P19(8) than L-P19(9/B). In a study of the systemic antimycotic activity of P19(8) in a mouse protection model, a modest prolongation in survival of Candida albicans-infected animals after intravenous administration was observed at 5 mg/kg peptide but not at higher doses. The implications of these observations for the systemic use of this type of peptide are discussed.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gene-encoded, ribosomally synthesized antimicrobial peptides (AMPs) are an important component of the innate immune response of eukaryotic organisms ranging from plants to invertebrate and vertebrate animals.15 Many AMPs act directly by permeabilizing the membrane of microbial targets, with a mode of action that does not appear to depend on specific receptors and which is thus not affected by the resistance mechanisms shown by a growing number of pathogens towards conventional antibiotics.3,6,7 Some endogenous AMPs have also been found to stimulate cell-mediated immunity by recruiting leucocytes.810 A considerable effort has been made to develop some classes of AMPs as pharmaceutical agents, mostly for the treatment of external local infections such as oral mucositis associated with chemotherapy, diabetic ulcers, chronic lung infections associated with cystic fibrosis, and ocular and bucco-dental infections, but none have as yet reached clinical use.1114 Although many AMPs have potent and broad-spectrum antimicrobial activity in vitro, their usefulness depends on various other factors. As they are generally cationic and amphipathic molecules, they can bind to host components such as extracellular proteins, lipoproteins, anionic constituents of cellular surfaces and of the extracellular matrix, and host cellular membranes themselves, which reduces bioavailability.1,15 Moreover, some of these peptides show harmful side effects, such as lysis of red blood cells at active antimicrobial concentrations.1618 Thus, although there are some indications that AMPs could also be used to treat systemic infections, a more detailed understanding of how they could affect host cells is called for.

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 {alpha}-helical AMPs on mouse primary cultures of resting or activated lymphocytes. These compounds were designed as part of a detailed structure–activity 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peptide design, synthesis and characterization

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 {alpha}-helical peptides, as described previously.20 Solid-phase syntheses were carried out on an automated synthesizer (Pioneer PerSeptive Biosystems) using PEG-PS resins (0.17–0.22 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 150–300 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 ({varepsilon}280 = 1290 M–1·cm–1). Circular dichroism (CD) spectra were obtained on a Jasco J-700 dichrometer using 2 mm path-length cuvettes and peptide concentrations of 4 x 10–5 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 ([{theta}]meas – [{theta}]rc)/([{theta}]{alpha} – [{theta}]rc), where [{theta}]meas is the measured ellipticity at 222 nm, [{theta}]rc is the ellipticity for unstructured peptide in the absence of additives (generally close to zero) and [{theta}]{alpha} is the ellipticity of a fully structured helix of length n, calculated using the relation [{theta}]{alpha} = 39 000 (1–4/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 RPMI–5% 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 (4–160 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 PBS–NaN3–bovine 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 8–10 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/Newmann–Keuls test for analysis of variance (ANOVA), whereas animal survival data were subjected to the non-parametric Kaplan–Meier test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In Table 1 we summarize the physicochemical characteristics and haemolytic activities of three artificial amphipathic, {alpha}-helical peptides. L-P19(9/B) and D-P19(9/B) have the same sequence, but the first is composed of L-amino acids while the second is composed of D-amino acids. They were designed to have a high charge (+9), a reasonable amphipathicity (relative hydrophobic moment of 0.6) and include three Aib residues to favour helix formation. Although they respectively form a right-handed and left-handed helix in the presence of trifluoroethanol (TFE) or SDS micelles (Figure 1), they show the same degree of structuring (~60% estimated helix content). P19(8) also has a high net charge (8+) but a lower tendency to structure (the helical content is ~40% in 50% TFE or SDS micelles; see Figure 1 and Table 1), due to the absence of Aib residues and the presence of glycine residues at positions 7 and 14, respectively, replacing an Aib and a norleucine (Nle) residue. None the less, it is slightly more hydrophobic than P19(9/B), as the other Aib residues are replaced by Nle.


View this table:
[in this window]
[in a new window]
 
Table 1.  Sequences and properties of artificial peptides
 


View larger version (18K):
[in this window]
[in a new window]
 
Figure 1. CD spectra of L-P19(9/B), D-P19(9/B) and P19(8) in the presence of 10 mM SDS. The inset shows the degree of helicity (% helix content) for L-P19(9/B) and P19(8) at increasing proportions of TFE.

 
To evaluate the effect of these AMPs on circulating blood cells, we determined the haemolytic activity at 10 and 100 µM (Table 1) by quantifying haemoglobin release and damage to mouse leucocytes from a spleen primary cell culture, and in particular B- and T-lymphocytes, after exposure to peptides at concentrations ranging from 4 to 160 µM, using flow cytometric methods (Tables 24). In an initial study, membrane permeabilization was shown by PI uptake in resting cells (Figure 2 and Table 2). Both P19(8) and D-P19(9/B) showed a membrane permeabilizing effect, with the appearance of a population of cells characterized by stronger fluorescence (see inset to Figure 2c with respect to that in a) and by a smaller size than that of untreated cells (lower forward scattering population in Figure 2b and c), although exposure to P19(8) resulted in a more limited effect than D-P19(9/B) under the same conditions (Figure 2 and Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2.  Percentage of PI-excluding cells following in vitro treatment with peptide for 30 or 60 min
 

View this table:
[in this window]
[in a new window]
 
Table 4.  Fraction of PI-excluding T-lymphoblasts following in vitro treatment with AMPs
 


View larger version (11K):
[in this window]
[in a new window]
 
Figure 2. Contour plots of lymphocytes treated with antimicrobial peptides. (a) Control; (b) P19(8) at 16 µM; (c) D-P19(9/B) at 16 µM. The insets show the fluorescence monoparametric histograms for regions highlighted in grey, which correspond, respectively, to viable cells in (a), and damaged, PI-permeable cells in (c). FS, forward scattering; SS, side scattering.

 
The morphological changes observed by flow cytometry correlate with those observed by SEM, in cells treated with P19(8) under the same conditions (Figure 3). Untreated leucocytes have a homogeneous morphology and undamaged membranes, as indicated by a single peak in the 3D density plot (Figure 3a, left panel), a relatively smooth surface in SEM images (Figure 3a, right panel) and a low PI uptake (inset to Figure 2a). Exposure to higher concentrations of the peptide resulted in a second peak appearing in the 3D density plots (Figure 3b–d, left panels), corresponding to a cell population with a smaller size (decreased forward scattering, FS) and increased fluorescence, indicative of membrane damage. After exposure to 16 µM peptide for 30 min, the majority of cells still belong to the ‘viable’ cell population, with unaltered size and low fluorescence, although SEM images show that a significant alteration of the cell surface occurs, with extensive blebbing. The smaller sized population was instead significant after exposure to 40 µM peptide and dominant at 160 µM peptide, the highest dose used (Figure 3c and d, left panels). SEM of analogously treated cells indicated the appearance of some completely collapsed cells at 40 µM, alongside apparently integral cells (Figure 3c, right panel). At 160 µM peptide most of the cells observed by SEM were collapsed, having lost any three-dimensional structure (Figure 3d, right panel), although a small proportion of cells with a less affected morphology were also present. The proportion of collapsed to integral cells at this concentration estimated by SEM is in qualitative agreement with the number of ‘viable’ cells obtained by flow cytometric estimation of PI exclusion (~20%, Table 2).



View larger version (59K):
[in this window]
[in a new window]
 
Figure 3. Three-dimensional density plot analysis (left) and SEM (right) of leucocytes treated with P19(8) at increasing concentrations. (a) Control; (b) 16 µM peptide; (c) 40 µM peptide; (d) 160 µM peptide. The black arrows indicate peaks corresponding to viable cells (V) and cells with damaged membranes (D). White arrows indicate collapsed lymphocytes.

 
A quantitative analysis of the effect of exposure to P19(8) and D-P19(9/B) on the percentage of viable cells, based on the FS signal and PI exclusion capabilities, is reported in Table 2. Damage to lymphocytes does not appear to be statistically significant up to 16 µM peptide for P19(8). A moderate cytotoxicity was instead observed at 40 µM, while most cells were damaged at 160 µM. These effects were evident after 30 min challenge and did not vary significantly after longer exposure. D-P19(9/B) was more cytotoxic at equivalent concentrations, and this effect was time dependent, as a substantial increase in damaged cells was observed after 1 h, even at the lowest concentration used (4 µM). These results parallel the haemolytic activity of the peptides (Table 1), indicating that D-P19(9/B) is generally more cytotoxic than P19(8).

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 30–60 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).


View this table:
[in this window]
[in a new window]
 
Table 3.  Evaluation of the effect of P19(8) on the cell cycle and on total protein content by dual FITC/PI staininga on surviving cells of primary lymphocyte cultures 24 h after exposure to peptide for 30 or 60 min
 
During systemic use of peptides, they are likely to encounter immune cells activated by the presence of an infective agent. As damage to the cell membrane appears to be an important aspect of their cytotoxic activity, this could be affected by changes in immune cell membrane properties that occur after stimulation.2628 In vitro activation of splenocytes was effected by treatment with ConA, and the resulting T-lymphoblasts were characterized by their S-phase activation, their phenotypes (CD3, CD4 and CD44) and their mitochondrial respiration as assessed using the JC1 probe. Table 4 reports the effect of exposure to P19(8), L-P19(9/B) and D-P19(9/B), for 30 or 60 min. Again, P19(8) causes the least damage, significantly reducing the percentage of viable cells only at 40 µM, in a time-independent manner. Results are similar to those obtained with resting cells (Table 2), suggesting that its cytotoxicity does not depend on the state of cell activation. D-P19(9/B) confirmed a greater and time-dependent toxicity, which was more marked for activated than for resting cells. L-P19(9/B) was the most cytotoxic for ConA-activated cells; the effect at 4 µM being comparable to that of D-P19(9/B) at 16 µM and to that of P19(8) at 40 µM. These results, combined with the haemolytic activity, confirm that the cytotoxicity increases in the order P19(8) < D-P19(9/B) < L-P19(9/B).

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 80–300 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.



View larger version (18K):
[in this window]
[in a new window]
 
Figure 4. Survival plot according to the Kaplan–Meier test, for mice treated iv with P19(8) 6 h after inoculation with 5 x 105 C. albicans cells. Results are based on 18 mice for the treated group and 15 mice for the control group. P = 0.044 according to the Mantel–Heaenzel log-rank test.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The systematic variation of several physicochemical parameters in {alpha}-helical AMPs, designed using a rationally derived sequence template,3,20,29,30 has led to the development of peptides such as L-P19(9/B), which has potent and broad-spectrum antimicrobial activity. This results from a combination of optimized characteristics, including high charge and amphipathicity, and a stabilized {alpha}-helical structure, but unfortunately is also accompanied by a relatively high haemolytic activity (Table 1). Modulation of helical structuring in P19(8) resulted in a strong reduction of haemolysis, albeit accompanied by a moderate reduction in antimicrobial potency. A more drastic inhibition of helix formation, obtained by insertion of proline or D-amino acids, severely limits antimicrobial activity as well.20 Another type of structural modification, formation of a left-handed helix in an all-D analogue of P19(9/B), also somewhat reduced haemolysis (Table 1), but in this case without significantly affecting antimicrobial activity.20

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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-helical AMPs.

From our study, we conclude that the in vitro cytotoxicity of the linear {alpha}-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
 
We are grateful to Dr D. Romeo for stimulating this work and critically reading the manuscript. We thank Mr F. Micali for advice with SEM sample preparation. The free availability of the flow cytometry facility of the Fondazione Callerio ONLUS is also gratefully acknowledged. This research was in part supported by Grants from the Italian Ministry of the Universities and Scientific Research (PRIN MM05265243) and the EU PANAD project QLRT-2000-00411. M. Bacac is supported by a grant from the Fondazione C&D Callerio ONLUS, Trieste, Italy.


    Footnotes
 
* Corresponding author. Tel: +39-040-5583529; Fax: +39-40-577435; E-mail: pacorsab{at}units.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Andreu, A. & Rivas, L. (1998). Animal antimicrobial peptides, an overview. Biopolymers 47, 415–33.[ISI][Medline]

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, 8856–61.[Abstract/Free Full Text]

3 . Tossi, A., Sandri, L. & Giangaspero, A. (2000). Amphipathic, alpha-helical antimicrobial peptides. Biopolymers 55, 4–30.[ISI][Medline]

4 . Tossi, A. & Sandri, L. (2002). Molecular diversity in gene- encoded, cationic antimicrobial polypeptides. Current Pharmaceutical Design 8, 743–61.[ISI][Medline]

5 . Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature 415, 389–95.[ISI][Medline]

6 . Matsuzaki, K. (1999). Why and how are peptide–lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochimica et Biophysica Acta 1462, 1–10.[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, 55–70.[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, 1069–74.[Abstract/Free Full Text]

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, 978–89.[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, 454–7.[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, 88–98.[ISI][Medline]

12 . Hancock, R. E. W. (2000). Cationic antimicrobial peptides, towards clinical applications. Expert Opinion on Investigational Drugs 9, 1723–9.[ISI][Medline]

13 . Lamb, H. M. & Wiseman, L. R. (1998). Pexiganan acetate. Drugs 56, 1047–52.[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, 216–22.[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, 22445–51.[Abstract/Free Full Text]

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, 109–14.[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, 3718–24.[Free Full Text]

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, 58–62.[ISI][Medline]

19 . Fromtling, R. A. (1998). Human mycoses and current antifungal therapy. Drug News Perspectives 11, 185–91.[ISI]

20 . Giangaspero, A., Sandri, L. & Tossi, A. (2001). Amphipathic {alpha}-helical antimicrobial peptides, a systematic study of the effects of structural and physical properties on biological activity. European Journal of Biochemistry 268, 5589–600.[Abstract/Free Full Text]

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, 3350–9.[ISI][Medline]

22 . Gobbo, M., Bioni, L., Filira, F., Formaggio, F., Crisma, M., Rocchi, R. et al. (1998). Helix induction potential of N-terminal {alpha}-methyl, {alpha}-amino acids. Letters in Peptide Science 5, 105–7.[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. 23–86. DHHS Publications, National Institutes of Health, Bethesda, MD, USA.

25 . Polak, A. (1998). Experimental models in antifungal chemotherapy. Mycoses 41, 1–30.[ISI][Medline]

26 . Lewis, R. S. & Cahalan, M. D. (1995). Potassium and calcium channels in lymphocytes. Annual Reviews in Immunology 13, 623–53.[ISI][Medline]

27 . MacDonald, R. & Nabholz, M. (1986). T-cell activation. Annual Reviews in Cell Biology 2, 231–53.

28 . Tossi, A., Tarantino, C. & Romeo, D. (1997). Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity. European Journal of Biochemistry 250, 549–58.[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, 202–6.[ISI][Medline]

30 . Risso, A., Zanetti, M. & Gennaro, R. (1998). Cytotoxicity and apoptosis mediated by two peptides of innate immunity. Cellular Immunology 189, 107–15.[ISI][Medline]

31 . van’t 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, 163–9.[ISI][Medline]