Published online before print October 21, 2005
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* Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy; and
Microbiology Section, Department of Pathology and Laboratory Medicine, University of Parma, Italy
1 Correspondence: Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail: vecchiar{at}unipg.it
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Key Words: killer mimotopes • antimicrobial peptides • antifungal compounds
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Despite the experimental data about the fungicidal and therapeutic activity of KP, nothing is known about its possible interaction with host cells. Indeed, should KP bind to selected cell populations (i.e., cells of the immune system), it could possibly modulate their functions and affect the outcome of infection. In this study, we investigated the ability of KP to bind to different murine immune cell populations and analyzed its effect on specific DC parameters.
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Cell separation
Dendritic cells (DC) and CD4+ T lymphocytes were separated from spleens of inbred BALB/c mice (Harlan Nossan Laboratories, Milan, Italy) using N-418 or L3T4 mAb-conjugated MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and magnetic separation was performed according to the manufacturer’s instructions. Peritoneal neutrophils or macrophages were collected 18 h or 4 days, respectively, after the intraperitoneal injection of 1 ml endotoxin-free 10% thioglycolate solution (Difco, Detroit, MI).
Cytofluorimetric analysis
For KP binding to cell-surface molecules, 1 x 106 DC, CD4+ T lymphocytes, macrophages, or neutrophils were incubated for 20 min at 37°C with 10 µg b-KP or b-SP in 1 ml RPMI 1640 with L-glutamine and 10% fetal calf serum (FCS; complete medium). After incubation, cells were reacted with avidin-fluorescein isothiocyanate (FITC), washed twice in fluorescence buffer [FB; phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and 0.4% sodium azide], and analyzed by FACScan (BD Biosciences, Franklin Lakes, NJ). To stain the total pool of KP, the cells were permeabilized for 10 min at room temperature with PBS containing 0.1% saponin (Sigma Chemical Co., St. Louis, MO), fixed in 1% paraformaldehyde, washed twice in FB containing 0.1% saponin and 5% FCS, and analyzed cytofluorimetrically as above. The intracellular KP was calculated by subtracting surface-bound KP from the total pool. As a control, b-SP was used. On cytofluorimetric analysis, nonviable cells were excluded by accepted procedures involving propidium iodide and narrow, forward-angle, light-scatter gating. Cells reacting directly with avidin-FITC were used to obtain background fluorescence values. Data are expressed as mean fluorescence intensity (MFI) of labeled cells.
For dose dependency of KP binding, DC were incubated for 20 min with 2, 5, 10, and 20 µg b-KP/106 cells, and for time dependency, 106 cells were incubated for different times with 10 µg b-KP. Following incubation, the cells were analyzed as reported above. Data are expressed as MFI or as percentage of labeled cells.
Fluorescence microscopy
In selected experiments, DC were incubated with b-KP or b-SP (10 µg/106 cells) for 20 min at 37°C in complete medium, reacted with avidin-FITC, washed, and examined under fluorescent light microscopy.
KP binding to DC and effect on activatory or costimulatory molecules expression
DC (106/ml) were incubated with anti-major histocompatibility complex (MHC) class I [2 µg, clone 2G5, mouse immunoglobulin G (IgG)2b, Serotec, Oxford, UK], anti-MHC class II (2 µg, clone IBL-5/22, rat IgG, Chemicon Int., Temecula, CA), anti-CD16/32 (2 µg, clone 93, rat IgG2b, Chemicon Int.), and anti-specific intercellular adhesion molecule-grabbing nonintegrin (SIGN) R1 (2 µg goat IgG, R&D Systems, Minneapolis, MN) antibodies in complete medium for 20 min at 37°C in 5% CO2. Subsequently, 10 µg b-KP or b-SP was added for a further 20-min incubation. After incubation, the cells were collected by centrifugation, fixed in 10% formalin, labeled with avidin FITC (40 min at 4°C), washed twice in FB, and analyzed by flow cytometry.
For analysis of activatory or costimulatory molecule surface expression, purified DC (106/ml) were incubated with or without KP or SP (10 µg/106 cells) in complete medium for 20 min at 37°C in 5% CO2. After incubation, the cells were collected by centrifugation; fixed in 10% formalin; reacted for 40 min at 4°C with labeled mAb to CD86 (10 µl, clone GL1, rat IgG2a), CD80 (10 µl, clone 16-10A1, hamster IgG), CD8
(10 µl, clone KT15, rat IgG2a), CD40 (10 µl, clone 1C10, rat IgG2a, all from Chemicon Int.), MHC class I (1 µg, clone SF1-1.1, mouse IgG2a), and MHC class II (1 µg, clone AMS-32.1, mouse IgG2b, all from BD Biosciences); washed twice in FB; and analyzed by flow cytometry. Matched antibody isotypes were used as negative controls.
Proliferation assay
DC (1x105) and CD4+ T lymphocytes (1x106) were purified from spleens and cocultured in round-bottom, 96-well plates in complete medium (final volume, 200 µl) in the presence or absence of anti-CD3 antibodies (0.2 µg, clone 17A2, Chemicon Int.) or phytohemagglutinin (PHA; 5 µg/ml, Sigma Chemical Co.), without or with 2 µg KP. Cells were cultured for 4 days at 37°C, 5% CO2. Eighteen hours before harvesting, cells were pulsed with 0.5 µCi [3H]-thymidine per well. Incorporation into cellular DNA was measured by liquid scintillation counting. The results are expressed as mean counts per minute (cpm) ± SEM of triplicate cultures.
Statistical analysis
Statistical analysis was performed with the Primer of Biostatistics software program. Data are reported as the mean ± SEM from replicate experiments. Data were evaluated by one-way ANOVA. Comparisons were done with unpaired student’s t test. A value of P < 0.05 was taken as significant.
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Figure 1. KP binds to APC. For evaluation of surface binding of KP (solid bars) or SP (hatched bars), 1 x 106/ml DC, macrophages (M ), neutrophils (PMN), or CD4+ T lymphocytes were incubated with 10 µg b-KP or b-SP for 20 min at 37°C in 5% CO2. For intracellular uptake of KP (open bars) or SP (dotted bars), cells were permeabilized with saponin and incubated with b-KP or b-SP. After addition of avidin-FITC, cells were analyzed by flow cytometry, and peptide binding and uptake were determined as described in Materials and Methods. Data are expressed as MFI of labeled cells.
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70% of cells recognized the peptide (Fig. 2B)
; after this time, KP was degraded/released, as manifested by the rapid decrease of percentage of positive cells (Fig. 2B)
. A negligible binding of SP was observed (Fig. 2B) . Further analysis of KP interaction with DC evidenced that the peptide was internalized, and the maximum uptake was observed within 20 min (Fig. 2C)
. Furthermore, KP surface binding and internalization were inhibited when the assay was performed in the presence of 20 mM EDTA or after incubation at 4°C (not shown). Figure 2D
shows the intense fluorescence of DC treated for 20 min with an optimal dose of KP and significantly less evident fluorescence observed with SP.
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Figure 2. KP binds to DC in a dose- and time-dependent manner. (A) For determination of dose dependency of KP binding, 1 x 106/ml DC were incubated with 2, 5, 10, and 20 µg b-KP for 20 min at 37°C in 5% CO2. As negative control, DC were also incubated with identical concentrations of b-SP. After addition of avidin-FITC, cells were analyzed by flow cytometry, as described in Materials and Methods. Data are expressed as MFI of labeled cells. (B and C) For time dependency, 1 x 106/ml DC were incubated for different times with 10 µg b-KP or b-SP at 37°C in 5% CO2. After addition of avidin-FITC, cells were analyzed by flow cytometry. Data are expressed as percentage of positive cells (B) or as MFI (C). The MFI values of surface binding (solid bars) and intracellular uptake (hatched bars) were calculated as described in Materials and Methods. The percentage of positive cells of DC treated with b-SP was less than 10%, and the MFI values ranged between 10 and 15 in all experiments performed. (D) DC, 1 x 106/ml, were incubated with b-KP or b-SP reacted with avidin-FITC, and subsequently examined under fluorescent light microscopy. Note the green fluorescence of KP-treated DC.
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R) the ability to recognize and interiorize different antigenic structures. In addition, the capability of MHC to directly bind some peptides from the extracellular compartment has been described [13
]. Given that KP is bound and internalized by DC, we considered C-type lectins, FcR, and MHC class I and class II as potential receptors for KP. To this end, KP binding was evaluated by cytofluorimetric analysis after blocking with specific antibodies, different cell-surface molecules including MHC class I, MHC class II, and CD16/32, and potential cellular receptors recognized by anti-SIGN R1 antibodies. Preliminary experiments to sort out the optimal dose of antibodies to block KP uptake were performed. Doses ranging from 1 to 5 µg were used for MHC class I, class II, CD16/32, and SIGN R1. We found that the doses ranging from 2 to 5 µg were necessary to obtain the maximum inhibition of KP uptake. Data, by using 2 µg antibodies, are reported. The results show that blocking of MHC class II, CD16/32, and cellular receptors reacting with anti-SIGN R1 antibodies, but not MHC class I, resulted in a significant decrease in KP binding, thus suggesting that KP could possibly engage several surface receptors (Fig. 3 ).
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Figure 3. KP binding to DC is impaired by selected mAbs. DC (1x106/ml) were incubated with anti-MHC class I, anti-MHC class II, anti-CD16/32, or anti-SIGN R1 antibodies (Abs; hatched bars) and with their matched isotypes (shaded bars) in complete medium for 20 min at 37°C in 5% CO2, and subsequently, 10 µg b-KP was added for a further 20-min incubation. Binding of b-KP (solid bars) and b-SP (open bars) to untreated cells is also reported. After addition of avidin-FITC, cells were analyzed by flow cytometry. Data are expressed as MFI of labeled cells. For details, see Materials and Methods. *,P < 0.01; **, P < 0.05, KP-treated versus controls, according to Student’s t-test.
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, CD80, and CD40, and it decreased the expression of CD86 and MHC class I and class II molecules (Fig. 4A
). However, despite the significant up-regulation of MFI for the above molecules, the percentage of positive cells did not manifest significant changes, suggesting that KP treatment does not induce phenotypic conversion of DC. Incubation with SP did not result in relevant modulation of costimulatory molecules expression (data not shown). To assess if KP, together with phenotypic changes, would also modulate the instructive antigen-presenting function of DC, KP-treated DC were cocultured with CD4+ T lymphocytes for 3 days in the presence or absence of anti-CD3 mAb or PHA, and lymphoproliferation was determined as [3H]-tymidine uptake. The results showed that treatment with KP significantly increased a CD4+ T cell blastogenic response (Fig. 4C)
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Figure 4. KP modulates DC phenotype and function. (A) For evaluation of CD8- or MHC class I expression, purified 1 x 106/ml DC were incubated with (open bars) or without (solid bars) 10 µg KP in complete medium for 20 min at 37°C in 5% CO2. After incubation, cells were reacted with R-phycoerythrin (RPE)-conjugated mAb to CD8- or MHC class I and analyzed by flow cytometry. For each molecule, data are expressed as MFI of labeled cells (left) and as a histogram (right). For details, see Materials and Methods. *, P < 0.01, KP-treated versus controls, according to Student’s t-test. (B) For evaluation of surface molecule expression, purified 1 x 106/ml DC were incubated with (open bars) or without (solid bars) 10 µg KP in complete medium for 20 min at 37°C in 5% CO2. After incubation, cells were reacted with RPE-conjugated mAb to CD80, CD86, CD80, and MHC class II and analyzed by flow cytometry. Data are expressed as MFI of labeled cells. For details, see Materials and Methods. *, P < 0.01, KP-treated versus controls, according to Student’s t-test. (C) For evaluation of the capacity of KP-treated DC to induce lymphocyte proliferation, 1 x 105 DC were cocultured with 1 x 106 CD4+ T lymphocytes in round-bottom, 96-well plates (final volume, 200 µl), unstimulated (solid bars) or in the presence of anti-CD3 antibodies (open bars) or PHA (hatched bars), alone or with 2 µg KP. Cells were cultured for 4 days at 37°C in 5% CO2. Eighteen hours before harvesting, cells were pulsed with 0.5 µCi [3H]-thymidine per well, and incorporation into cellular DNA was measured by liquid scintillation counting. The results are expressed as mean cpm ± SEM of triplicate cultures. *, P < 0.01, KP-treated versus controls, according to Student’s t-test.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The wide antimicrobial spectrum of KP activity has been demonstrated in a variety of studies. In particular, strong microbicidal activity has been detected in vitro and/or in vivo against fungi [9 10 11 ] and other multidrug-resistant eukaryotic and prokaryotic pathogenic microorganisms [15 ]. The direct microbicidal activity of KP has been associated with its binding to ß 1,3-glucan on the surface of many microorganisms, being neutralized by laminarin, a soluble ß 1,3-glucan [9 , 10 ]. Although the direct microbicidal and related therapeutic effect has been studied in depth, no information currently exists about the interaction of KP with immune cells and therefore, about the possibility that the beneficial effects observed in vivo could also be a result of its immunomodulating activity. In this report, we evidenced for the first time an interaction of KP with immune cells, demonstrating that this decapeptide specifically binds to cells of the monocyte-macrophage lineage. In particular, KP binds to a high level on DC, to a lesser extent on macrophages, and does not interact with neutrophils and T cells.
The capacity of KP to distinguish among different immune cells was unexpected and suggests the engagement of specific molecules on the cell surface. Indeed, KP reacted with MHC class II, expressed to a high level on DC and to a lesser extent on macrophages and with DC surface molecules recognized by anti-mouse SIGN R1 antibodies. Conversely, we found only a marginal engagement of CD16/32 expressed on neutrophils [16 ] and lack of engagement of MHC class I, constitutively expressed on neutrophils and T cells. Thus, KP binds specifically to DC and macrophages through SIGN R1 and in a second instance, MHC class II cellular receptors.
It has been reported that SIGN R1 recognizes different molecules, such as specific carbohydrate structures present on pathogens as well as lectin-specific antibodies [17 ]. There is no evidence so far that antibodies to SIGN R1 react with a lectin on the DC surface, and we do not know with what exactly the antibody reacts; however, an involvement of cellular molecules recognized by SIGN R1 in KP uptake clearly occurs. As MHC class II can bind some peptides from the extracellular compartment [13 ], it is conceivable the KP surface detection could be partially a result of direct MHC class II engagement.
DC are key initiators of a primary immune response, being professional APC, and have an outstanding ability to prime naïve T cells [18
, 19
], driving the ensuing response into T helper cell type 1 (Th1) or Th2 [20
21
22
]. KP modulated the expression of costimulatory and MHC class I and class II molecules on DC. In particular, strong up-regulation of CD40 and down-regulation of CD86 were observed. As the development of acquired immune response is dependent on the signaling of CD40 by CD40 ligand [23
24
25
26
], which is in turn inhibited by CD86 [27
], it is possible to speculate that the beneficial effect of KP in experimental models of infection could be partially a result of the elicitation of an optimal, antimicrobial, cell-mediated immune response. This is in line with the finding that KP induced an increased expression of CD8- on DC, and CD8- positive DC are considered predominant producers of interleukin-12, a key cytokine for induction of a protective Th1 response against many pathogens, including fungi [28
29
30
]. However, despite the significant up-regulation of MFI for CD8-, the percentage of positive cells did not manifest any significant change, suggesting that KP treatment does not induce phenotypic conversion of DC during 20 min of incubation. The phenotypic changes of DC occur immediately after KP addition. Conversely, the maximum KP uptake is evidenced within 20 min, and a comparable time for DC activation was reported for lipopolysaccharide and recombinant proteins [31
].
We found that KP enhanced CD80 and decreased CD86 expression on DC. At present, it is not completely clear whether CD80 or CD86 will promote or inhibit T cell responses. The current view is that CD86 is the initial view costimulatory ligand, and CD80 has a role following APC activation [27 ]. Recently, an alternative has been described, in which CD80 is the initial ligand responsible for maintaining aspects of immune tolerance through interactions with CD152. These inhibitory functions can then be overridden by the up-regulation of CD86 on DC as a result of inflammatory stimuli, leading to immune activation [27 ]. Thus, enhancement of CD80 expression by KP could be important in activation and/or modulation of T cell reactivity.
The ability of KP-treated DC to induce T cell activation upon anti-CD3 stimulation suggests that the observed reduction of MHC molecules could not play a crucial role in some experimental conditions. It is interesting that KP was found to inhibit the expression of MHC class I and MHC class II. Although the drastic decline of MHC class II could likely be ascribed to the capacity of KP to engage this molecule directly, a real down-regulation could occur for MHC class I, unable to bind KP. Then again, the reduction of MHC class I and class II does not necessary imply that antigen presentation is impaired, as this process is a result of complex mechanisms that initiate in the endosome compartment with peptide delivery for rapid and efficient loading of MHC epitopes [32 , 33 ].
In conclusion, our results evidence that the selective interaction of KP with DC results in the modulation of cell phenotype and function. This mechanism could concur with KP direct fungicidal activity to the immunotherapeutic effect of KP in different experimental infections, suggesting a potential use of this decapeptide to selectively modulate antigen presentation and antimicrobial immune response.
Received February 24, 2005; revised May 24, 2005; accepted June 3, 2005.
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RII receptors Cell. Immunol. 201,89-93[CrossRef][Medline]+ and CD8– subclasses of dendritic cells direct the development of distinct T helper cells in vivo J. Exp. Med. 189,587-592This article has been cited by other articles:
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  G. Conti, W. Magliani, S. Conti, L. Nencioni, R. Sgarbanti, A. T. Palamara, and L. Polonelli Therapeutic Activity of an Anti-Idiotypic Antibody-Derived Killer Peptide against Influenza A Virus Experimental Infection Antimicrob. Agents Chemother., December 1, 2008; 52(12): 4331 - 4337. [Abstract] [Full Text] [PDF]
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