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Maturation of bovine dendritic cells by lipopeptides
Veterinary Immunology and Immunopathology 95 (2003) 21–31
Maturation of bovine dendritic cells by lipopeptides Jayne C. Hopea,*, Adam O. Whelanb, R.G. Hewinsonb, Martin Vordermeierb, Chris J. Howarda a
Institute for Animal Health, Compton, Newbury, Berkshire, UK b Veterinary Laboratories Agency, Addlestone, Weybridge, UK
Received 26 August 2002; received in revised form 16 April 2003; accepted 16 April 2003
Abstract The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge. The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells. We report here that bovine DC are also induced to mature following lipopeptide stimulation. Exposure of DC to the model lipopeptide Pam3CSK4 was associated with increased expression of MHC, costimulatory molecules, and enhanced secretion of IL-12 and TNFa. Lipopeptide-matured DC were superior in their ability to induce T cell activation and IFNg secretion. In contrast, exposure of MF to lipopeptides induced down-regulation of MHC expression and much lower increases in IL-12 secretion. A lipopeptide derived from the sequence of a relevant mycobacterial lipoprotein, MPB83, also influenced bovine DC by stimulating increases in IL-12 and TNFa secretion. These different changes in bovine DC and MF may have important implications for immune responses induced following bacterial infection with uptake of microbes by DC resulting in potentiation of their immunostimulatory capacity and uptake by MF having a much less marked effect on immune responses. # 2003 Elsevier B.V. All rights reserved. Keywords: Dendritic cells; Macrophages; Mycobacterium bovis; Lipopeptides
1. Introduction Dendritic cells are unique in their capacity to initiate immune responses. The effects of microbial products on these cells and the effect that these have on DC function have been studied extensively in human and mouse systems. Stimulation or infection of DC with microbes and their products is associated Abbreviations: DC, dendritic cells; TNF, tumour necrosis factor; MF, macrophages; TLR, toll-like receptor; IL, interleukin * Corresponding author. Tel.: þ44-1635-578411; fax: þ44-1635-577263. E-mail address: [email protected] (J.C. Hope).
with maturation of the DC such that their capacity to induce T cell activation is enhanced. These effects on DC are likely to lead to the induction of more efficient immune responses following microbial challenge. For mycobacteria, the lipoproteins expressed by the bacteria are thought to be largely responsible for the maturation events observed in DC (Brightbill et al., 1999; Hertz et al., 2001; Thoma-Uszynski et al., 2001). Thus, human DC infected with M. tuberculosis or M. bovis BCG show enhanced expression of MHC and costimulatory molecules, and also secrete enhanced levels of IL-12 and TNFa (Henderson et al., 1997; Demangel et al., 1999; Kim et al., 1999). This can be mimicked in vitro by the addition
0165-2427/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-2427(03)00104-1
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of the 19 kDa lipoprotein isolated from M. tuberculosis and also by synthetic lipopeptides that are similar in size and hydrophobicity to microbial lipoproteins (Hertz et al., 2001; Thoma-Uszynski et al., 2000). These effects on human DC have been attributed to events downstream of stimulation of toll-like receptor 2 (TLR-2; Hertz et al., 2001; Thoma-Uszynski et al., 2000). Other bacterial products can activate antigen presenting cells via other TLR, for example CpG motifs act via TLR-9 and LPS via TLR-4. The TLR family of receptors are evolutionary conserved receptors present most abundantly on antigen presenting cells in mammals, and are an important element of the innate response to infection (Medzhitov et al., 1997; Krutzik et al., 2001). Stimulation of TLR by microbial products is likely to be an important mechanism by which cells of the innate immune system can recognise and be activated by microbial products, leading to the initiation of an adaptive immune response. The effects of microbial products on macrophages (MF) have also been described and linked to TLR expression. One group has shown that these cells respond to lipopeptides and live M. tuberculosis by down-regulating MHC molecules, a mechanism which may allow immune evasion (Noss et al., 2000, 2001). As MF are considered the major host cell for mycobacteria in vivo their responses may also be pivotal in determining whether the outcome of infection is disease or immunity. Infection of cattle with M. bovis causes bovine tuberculosis, a problem of major concern which is becoming more common in UK cattle herds. Understanding the immune response that occurs following M. bovis infection in cattle will provide important information that might aid better vaccine design or the development of more effective control strategies. The central role of MF in mycobacterial infections is well established. Infection of bovine DC with M. bovis BCG has been reported and these cells are capable of inducing vigorous antigen specific T cell responses in vitro (Hope et al., 2000). The effects of infection with M. bovis or M. bovis BCG on DC maturation are similar to those reported for human DC infected with mycobacteria (Thom and Hope, unpublished data). In this study we have assessed the effect of lipopeptides on bovine DC and MF and we describe changes in a number of parameters that are important for immune regulation. This is the first report directly
comparing the effects of microbial lipopeptides on the phenotype and function of DC and MF derived from the same source and also in a relevant natural target species. We report that lipopeptides induce both functional and phenotypic maturation of DC and also affect MHC expression by MF.
2. Materials and methods 2.1. Lipopeptides The lipopeptide Pam3CSK4, where the Pam3C is represented by a palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl) residue, was obtained from EMC Microcollections, Tubingen, Germany. A lipopeptide based on the N-terminus of M. bovis MPB83 (Pam3CSS) was synthesised by solid phase F-moc chemistry. Initially, a di-amino acid peptide S–S was synthesised on a Model 432 peptide synthesiser (PE-Applied Biosystems, Warrington, UK). Pam3C (Bachem, St. Helens, UK) was dissolved in dichloromethane and then coupled to the amide group of the S–S resin attached peptide in the presence of 2-(1H-benzotriazyl-1-yl)1,1,3,3-tetramethyluranium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA) for 14 h at 20 8C (reagents supplied by PE-Applied Biosystems). The molar ratios of the S–S peptide, Pam3C, HBTU and DIEA in the coupling reaction were 1:3:2.7:3, respectively. A further unmodified control peptide CSS was synthesised on a Model 432 peptide synthesiser. Cleavage of peptides from the resin was performed by TFA hydrolysis. The peptide CSS was recovered from the hydrolysis solution by precipitation in methyl-tert-butyl ether. The recovered peptide was re-suspended in water and lyophilised two consecutive times. Due to the lipophillic nature of the peptide Pam3CSS, the peptide was recovered from the hydrolysis solution by lyophilisation and then resuspended in water and lyophilised further two times. The identity of peptides was confirmed by electrospray mass spectrometry and purity confirmed to be >80% by reverse phase HPLC. The MPB83 lipopeptide Pam3CSS and control peptide CSS are very different in molecular weight (1048.7 and 294.3, respectively). Thus these were tested at equimolar concentrations to ensure accurate comparison of their immunomodulatory properties.
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For comparative purposes with the synthetic lipopeptide Pam3CSK4, 50 mM of Pam3CSS equates to a concentration of 55 mg/ml. All peptides were diluted for use in PBS containing 0.1% Tween and aliquots were stored at 20 8C. Immediately prior to use the peptides were thawed and briefly sonicated to create a uniform suspension. 2.2. Generation of bovine blood derived-dendritic cells and MF Bovine monocytes were cultured with GMCSF and IL-4 to derive DC by a slight modification of the method described previously (Werling et al., 1999). Peripheral blood mononuclear cells (PBMC) were derived by centrifugation over Histopaque (Sigma), and were incubated with anti-human CD14 labelled super-paramagnetic particles (Miltenyi-Biotech, Bergisch Gladbach, Germany), and labelled cells were isolated from a Midimacs column (Miltenyi-Biotech) according to the manufacturers instructions. The purity of the cells was evaluated by flow cytometry and shown in each case to be >98%. Cell viability was >95%. Cells were adjusted to 8 105 ml1 in RPMI-1640 medium containing Glutamax-1 (Life Technologies, Paisley, UK), 10% heat inactivated FCS, 5 105 M 2-ME, 50 mg/ml gentamycin (tissue culture medium; TCM), 200 U/ml COS-7 cell derived bovine rIL-4 (Hope et al., 2000) and 0.2 U/ml bovine rGMCSF (units based on induction of 1/2 maximal proliferation in bone marrow precursor cells) and 3 ml of this suspension was added per well of sixwell plates. After 3 days of culture, the peptides were added to give the indicated final concentrations and the DC were cultured for an additional 2 days. On day 5 of culture the DC were harvested and counted. MF were CD14þ cells isolated as described above and cultured for 3 days in IMDM (Life Technologies) containing 10% FCS, 5 105 M 2-Me and 50 mg/ml gentamycin. Peptides were added on day 3 of culture and the MF cultured for a further 2 days prior to analysis. 2.3. Flow cytometry The mouse anti-bovine mAb used were CC158 (anti-MHC class II; IgG2a), IL-A88 (anti-MHC
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class I; IgG2a), CC14 (anti-CD1b; IgG1), IL-A156, IL-A159, IL-A190 (anti-CD40, CD80 and CD86, respectively, all IgG1; provided by Dr. N. MacHugh, Centre for Tropical Medicine, Roslin, UK). Control mAb used within the study were AV20 (mouse IgG1) and AV37 (mouse IgG2a), that are directed against chicken bursal B cells, chicken CD4þ cells and chicken spleen cell subset, respectively, all provided by Dr. T.F. Davison, IAH. DC or MF were incubated with primary mAb at pre-determined optimal concentrations for 10 min, then washed extensively. Bound mAb was detected with FITC labelled anti-mouse IgG (Southern Biotechnologies, Birmingham, AL, USA). The cells were analysed on a FACSCalibur (Becton Dickinson) and immunofluorescent staining was analysed using Win-MDI software. 10,000 gated cells were analysed for each parameter. 2.4. Proliferation assays and measurement of IFNg Allogeneic PBMC (105 per well) were incubated in triplicate with 1 104 irradiated MF or DC (20 Gy from a 137 Cssource) in a total volume of 200 ml TCM. Cultures were incubated for 5 days at 37 8C. 37mBq [3 H]-thymidine (3 HTdR; DuPont, Stevenage, UK) was added for the final 18 h of culture. Results are expressed as mean S:D: of triplicate wells. In some experiments supernatants were harvested from parallel cultures on day 4 and IFNg was measured by ELISA as previously described (15). Results are expressed as pg/ml. 2.5. TNFa and IL-12 ELISA Levels of TNFa and IL-12 in culture supernatants derived from MF or DC were assessed by ELISA. Antibodies to TNFa and IL-12 were generated by immunisation of mice with recombinant bovine TNFa or IL-12, and hybridomas were generated according to a standard method (Kwong et al., 2002). Pairs of mAb were tested for suitability in ELISA and both recombinant and natural TNFa or IL-12 were shown to be detected in an ELISA using mAbs CC327 and biotinylated-CC328 for TNFa (unpublished data), or mAbs CC301 and biotinylated-CC326 for IL-12 (Hope et al., 2002). The IL-12 ELISA was performed using the protocol as described by Kwong et al. (2002).
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For the TNFa ELISA, black microplates (Porvair, Shepperton, UK) were coated with 2 mg/ml CC327 and incubated overnight at room temperature. The plates were washed in washing buffer and blocking buffer was added for 1 h. Following a further washing step, samples were added for 1 h. Dilutions of the standard preparation of COS-7 cell derived rbo TNFa (in blocking buffer) were also added to the plates for 1 h. Following washing CC328-biotin (1 mg/ml in blocking buffer) was added for 1 h, followed by washing and addition of SA-HRP for 45 min. Following the final washing step the substrate
(Super Signal ELISA femto maximum sensitivity substrate; Pierce) was added for 1 min and relative light units (RLU) were read on an Anthos LUCY 1.0 luminometer (Anthos Labtec, Salzburg, Austria). Units per millilitre were calculated in comparison with the standard. 2.6. Statistical analysis Statistical analyses were performed using a paired Student’s t-test. P-values of less than 0.05 were considered significant.
Fig. 1. Phenotypic analysis of DC exposed to lipopeptides. Dendritic cells were exposed to Pam3CSK4 at the indicated concentrations for 48 h. Expression of the indicated surface molecules was assessed by flow cytometry. The results are expressed as mean fluorescence intensity (MFI) of cells within the live cell gate. One representative experiment of four is shown.
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3. Results 3.1. Stimulation of bovine DC with lipopeptides induced increases in expression of CD40, CD80 and CD1 On day 3 of DC culture, the lipopeptide Pam3CSK4 was added. After a further 2 days of culture the DC were harvested and analysed by flow cytometry for the expression of MHC class I, MHC class II, CD40, CD80, CD86 and CD1b. Following exposure of DC to Pam3CSK4 we observed a dose-dependent increase in the level of CD40, CD80, CD86 and CD1b (Fig. 1). More variable effects on expression of MHC class I and MHC class II were observed. In some experiments expression was unaffected by treatment, whereas in others both MHC I and MHC II were increased following exposure of DC to lipopeptide (Fig. 1). No effects on binding of control mAb were observed under any conditions (data not shown). 3.2. IL-12 and TNFa secretion by DC following culture with lipopeptides Exposure of DC to Pam3CSK4 was associated with a dose-dependent increase in IL-12 secretion with effects observed after stimulation with as low as 0.1 mg/ml of lipopeptide (Fig. 2a). In addition, treatment of DC with Pam3CSK4 led to a dose-dependent increase in TNFa secretion (Fig. 2b). 3.3. Increased allogeneic stimulation of T cells following culture of DC with lipopeptides In a further set of experiments, the capacity of DC to induce proliferation and to produce IFNg after stimulation with allogeneic PBMC was assessed. Following culture of DC with Pam3CSK4 there was a small but significant increase in the capacity of DC to stimulate allogeneic PBMC (P < 0:05; data not shown). In addition, stimulation of allogeneic PBMC with DC cultured in the presence of Pam3CSK4 (¼0.1 mg/ml) significantly increased the secretion of IFNg (P < 0:05; Fig. 2c). 3.4. Effects of lipopeptides on bovine MF Monocytes were cultured for 3 days then exposed to Pam3CSK4 for an additional 48 h. The effects on
Fig. 2. Lipopeptide stimulated DC secrete IL-12 and TNFa and induce increased IFNg synthesis by allogeneic PBMC. Dendritic cells were exposed to Pam3CSK4 at the indicated concentrations for 48 h. Supernatants were taken and assayed for IL-12 (a) and TNFa (b) by ELISA. The results are shown as biological units per millilitre. For MLR 104 stimulated APC were added to 105 allogeneic PBMC (c). Supernatants were derived after 4 days of culture and assayed by ELISA for the presence of IFNg. Results are expressed as pg/ml. One representative experiment of four is shown.
CD40, CD80, CD86 and CD1 expression on MF were noted only at the highest concentration of lipopeptide tested (Fig. 3). These were paralleled by increases in non-specific binding of control mAb (data not shown). However, addition of lipopeptide to cultures of MF decreased the expression of MHC class I and MHC class II in a dose-dependent manner (Fig. 3).
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Fig. 3. Effect of lipopeptide exposure on bovine MF surface molecule expression. MF were exposed to Pam3CSK4 at the indicated concentrations for 48 h. Expression of the indicated surface molecules was assessed by flow cytometry. The results are expressed as MFI of cells within the live cell gate. One representative experiment of three is shown.
The capacity of MF to induce allogeneic MLR proliferation (data not shown) and IFNg responses (Fig. 4c) was not affected when the cells were exposed to Pam3CSK4. In addition, much lower levels of IL-12 and TNF secretion were induced upon lipopeptide stimulation of compared to DC. 3.5. Effects of M. bovis derived lipopeptide on bovine DC To assess whether lipopeptides derived from the sequences of mycobacterial lipoproteins could also elicit changes in bovine DC and MF, a lipopeptide was synthesised based on the N-terminus of MPB83, a major secreted lipoprotein of M. bovis.
Amino-terminal sequencing of a native form of MPB83 from a M. bovis culture supernatant previously identified the loss of the first three amino acids (C–S–S) from the mature form of the protein (Whelan et al., 2003). Since the site of lipid modification of MPB83 is predicted to be at the amino-terminal Cys residue of the mature protein (Hewinson et al., 1996), the synthesis of the peptide Pam3CSS represented a biological relevant mycobacterial lipopeptide. The lipopeptide or non-lipoylated control peptides were added to DC and IL-12 secretion was assessed after 48 h of culture. The MPB83-derived lipopeptide (Fig. 5, black bars) induced dose-dependent increases in IL-12 (Fig. 5a) and TNFa (Fig. 5b) secretion from DC that were significantly different from those induced
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Fig. 4. Effects of lipopeptide stimulation on MF cytokine secretion and MLR inducing capacity. MF were exposed to Pam3CSK4 at the indicated concentrations for 48 h. Supernatants were taken and assayed for IL-12 (a) and TNFa (b) by ELISA. The results are shown as biological units per milliliter. For MLR 104 stimulated APC were added to 105 allogeneic PBMC (c). Supernatants were derived after 4 days of culture and assayed by ELISA for the presence of IFNg. Results are expressed as pg/ml. One representative experiment of four is shown.
by the control peptide (P < 0:05; Fig. 5, open bars). In contrast stimulation of MF with the MPB83-derived lipopeptide did not induce IL-12 secretion (data not shown).
4. Discussion DC are unique in their capacity to activate naive lymphocytes, resulting in the initiation of immune
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Fig. 5. Effect of M. bovis lipopeptide on cytokine expression by bovine DC. Dendritic cells were exposed to the M. bovis MPB83 lipopeptide Pam3CSS at the indicated concentrations for 48 h. Supernatants were taken and assayed for IL-12 (a) and TNFa (b) by ELISA. The results are shown as biological units per milliliter. One representative experiment of two.
responses. A number of studies have suggested that microbial products, including lipopeptides and lipoproteins, may act upon DC to enhance their antigen presenting and costimulatory capacity (Hertz et al., 2001; Thoma-Uszynski et al., 2001). Stimulation of DC with mycobacteria increases expression of MHC and costimulatory molecules, as well as enhancing their secretion of immunoregulatory cytokines such as IL-12 (Henderson et al., 1997; Demangel et al., 1999). We have demonstrated that culture of immature bovine DC with synthetic lipopeptides increases cell surface expression of CD40, CD80, CD86 and CD1, resulting in cells with increased T cell-stimulatory capacity when compared to cells cultured without lipopeptide. Functionally, the lipopeptide-matured
DC have enhanced T cell-stimulatory activity in allogeneic MLR and can enhance IFNg synthesis by allogeneic PBMC. In addition, lipopeptide stimulated DC expressed increased levels of IL-12 and TNFa, consistent with maturation. Moreover, we have shown that a lipopeptide based on the M. bovis secreted protein MPB83 also induces these maturation events, providing evidence that the DC maturation that is observed upon infection with M. bovis (Hope, unpublished observations) is, at least in part, induced by lipopeptides secreted from the mycobacterium. Increases in secretion of IL-12 and TNFa, alongside an enhanced capacity to stimulate T cells present within PBMC may have important implications for the induction of effective immune responses against M. bovis.
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Previous studies have demonstrated that stimulation with LPS or with live bacteria induces DC maturation. Stimulation of immature DC with LPS from Escherichia coli (Sallusto and Lanzavecchia, 1994) induced increases in a number of cell surface markers, including MHC class II, CD40, CD80, CD54, and CD58, whereas expression of CD14, CD32, and endocytic activity was reduced. In addition, infection of human DC with live bacillus Calmette-Gue´ rin (Kim et al., 1999), M. tuberculosis (Henderson et al., 1997), or Listeria monocytogenes (Kolb-Maurer et al., 2000), results in an increase in MHC and costimulatory molecule expression and enhanced T cell-stimulatory activity. Similar results were obtained with murine DC exposed to M. tuberculosis. Following infection of murine DC, expression of MHC and costimulatory molecules was upregulated and these DC showed enhanced ability to induce T cell responses (Demangel et al., 1999). Studies by Hertz et al., demonstrated that the 19 kDa lipoprotein from M. tuberculosis, as well as synthetic lipopeptides, induced DC maturation. The resulting mature DC had increased cell surface expression of MHC class II, CD80, CD83, CD86, CD54, and CD58, suggesting that the lipopeptide alone is sufficient to induce maturation events (Hertz et al., 2001). Lipopeptide-matured DC were also more potent than immature DC in stimulating T cells in a MLR (Hertz et al., 2001). Together, these findings provide a mechanism by which cells of the innate immune system can recognise and be activated by microbial products, leading to the initiation of an adaptive immune response. The study described herein illustrates that bovine DC respond to lipopeptides in a similar way, by upregulating expression of MHC and costimulatory molecules, including cytokines. A lipopeptide from the M. bovis MPB83 protein also increased the secretion of immunologically relevant cytokines by bovine DC. These effects are likely to reflect those observed following infection of DC by mycobacteria. In preliminary studies we have shown that infection of DC with virulent M. bovis or M. bovis BCG does indeed enhance expression of IL-12, TNFa and surface molecules including CD40 and CD80 (Hope and Thom, unpublished results). In addition to the modulation of immune responses by the lipid moiety, we have data that allows us to hypothesise that the length of the amino acid sequence attached to the lipopeptide, or its hydrophobicity relative to the lipid
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moiety, might be important for the induction of DC maturation. Thus, the longer MPB83-derived lipopeptide Pam3CSSTKPVSQDTSPKPA did not induce IL-12 secretion or phenotypic changes in DC (data not shown), whereas the shorter and more hydrophobic lipopeptide Pam3CSS exhibited significant biological effects. The production of IL-12 by DC infected with microbial pathogens is likely to have important implications for the outcome of infection. IL-12 is known to one of the most important mediators determining Th1 bias of the immune response (Macatonia et al., 1993). Th1 biased immunity is regarded as being central to effective immune responses against a number of bacterial pathogens. In this study, lipopeptide-matured DC showed enhanced capacity to stimulate IFNg secretion from allogeneic PBMC indicating that there may be more effective stimulation of Th1 responses. The increased secretion of IL-12 by lipopeptidematured DC may also stimulate NK cells which may contribute to the secretion of IFNg. Upregulation of costimulatory molecules and MHC may also contribute to the induction of more effective immune cell activation. Taken together the result of lipopeptide exposure of DC is likely to be initiation of Th1 cell activation. In addition to effects on DC, synthetic lipopeptides derived from the N-terminus of bacterial lipoproteins of the kind we have used in this study have been described as potent activators of murine and human MF and monocytes and polyclonal stimulators of murine B cells (Bessler et al., 1997a,b). They are also efficient immunoadjuvants in parenteral, oral and nasal immunisation either in combination with, or after covalent linkage to an antigen. Enhanced antibody-mediated immune responses and enhanced cellular immune responses in vivo have been reported after vaccination with lipopeptide–antigen conjugates, including the induction of MHC class I restricted CTL (Bessler et al., 1997a,b; Wiesmuller et al., 1989; Deres et al., 1989). The present paper is, to our knowledge, the first study describing biological activities of synthetic bacterial lipopeptides on bovine leukocytes and provides important comparisons of the effects of lipopeptides on different types of antigen presenting cells. Interestingly, in contrast to studies in other species like mice and humans that demonstrated an effect of such lipopeptides on MF, e.g. TNF-a
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production (Hauschildt et al., 1990; Hoffmann et al., 1988), we could not demonstrate significant lipopeptide induced cytokine production (IL-12 or TNF-a) by bovine MF. The most notable effect on bovine MF observed in the present investigation was the downregulation of MHC expression following lipopeptide exposure. Other studies have reported similar effects on MHC expression in murine and human MF following exposure to lipopeptides or to mycobacteria (Noss et al., 2000; Hauschildt et al., 1990). The downregulation of MHC class II may be a mechanism by which mycobacteria resident within MF evade detection by reactive T lymphocytes thus enabling survival and perpetuation of the infection. Extending the in vitro data presented in the present study to in vivo responses, we recently demonstrated that co-injection into cattle of lipopeptide with a skin test antigen significantly enhanced the skin test response. In 4/7 M. bovis infected calves tested Pam3CSK4 enhanced the skin test reactivity against the test antigen ESAT-6 (Whelan et al., 2003). These results suggest that lipopeptides may serve as effective vaccine adjuvants in cattle as well as other species. The mechanism by which microbe-induced DC maturation occurs is mediated by toll-like receptors (TLR; Krutzik et al., 2001). TLRs are a family of at least 10 transmembrane proteins that are evolutionarily conserved in many species (Medzhitov et al., 1997). The result of signalling through TLR is induction of the NFkB signalling pathway and the production of cytokines (Brightbill et al., 1999). Lipopeptides and lipoproteins activate cells of the immune system in a TLR-2 dependent manner (Hertz et al., 2001; Thoma-Uszynski et al., 2000; Noss et al., 2001). We have recently shown that bovine DC express RNA for TLR-2 (Hope and Werling, unpublished data) suggesting that lipopeptides could signal through this pathway in the bovine system. This study illustrates that in M. bovis the lipopeptides are likely to be important in inducing events following infection with pathogenic bacteria and provides data regarding the effects of microbes on DC and MF that may be relevant to the pathogenesis of disease. References Bessler, W.G., Heinevetter, L., Wiesmuller, K.H., Jung, G., Baier, W., Huber, M., Lorenz, A.R., Esche, U.V., Mittenbuhler,
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J.C. Hope et al. / Veterinary Immunology and Immunopathology 95 (2003) 21–31 Kolb-Maurer, A., Gentschev, I., Fries, H.W., Fiedler, F., Brocker, E.B., Kampgen, E., Goebel, W., 2000. Listeria monocytogenes infected human dendritic cells: uptake and host cell response. Infect. Immun. 68, 3680. Krutzik, S.R., Sieling, P.A., Modlin, R.L., 2001. The role of toll like receptors in host defence against microbial infection. Curr. Opin. Immunol. 13, 104–108. Kwong, L.S., Hope, J.C., Thom, M.L., Sopp, P., Duggan, S., Bembridge, G.P., Howard, C.J., 2002. Development of an ELISA for bovine IL-10. Vet. Immunol. Immunopathol. 85, 213–223. Macatonia, S.E., Hsieh, C.S., Murphy, K.M., O’Garra, A., 1993. Dendritic cells and macrophages are required for Th1 development of CD4þ T cells from ab TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFNg production is IFNg dependent. Int. Immunol. 5, 1119–1128. Medzhitov, R., Preston-Hurlburt, P., Janeway, C.A., 1997. A human homologue of the Drosophila toll protein signals activation of adaptive immunity. Nature 388, 394. Noss, E.H., Harding, C.V., Boom, W.H., 2000. Mycobacterium tuberculosis inhibits MHC class II antigen processing in murine bone marrow macrophages. Cell. Immunol. 201, 63. Noss, E.H., Pai, R.K., Sellati, T.J., Radolf, J.D., Belisle, J., Golenbock, D.T., Boom, W.H., Harding, C.V., 2001. Toll-like receptor 2 dependent inhibition of macrophage class II MHC expression and antigen processing by 19 kDa lipoprotein of Mycobacterium tuberculosis. J. Immunol. 167, 910–918.
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Sallusto, F., Lanzavecchia, A., 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109–1118. Thoma-Uszynski, S., Kiertscher, S.M., Ochoa, M.T., Bouis, D.A., Norgard, M.V., Miyake, K., Godowski, P.J., Roth, M.D., Modlin, R.L., 2000. Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10. J. Immunol. 165, 3804–3810. Thoma-Uszynski, S., Stenger, S., Takeuchi, O., Ochoa, M.T., Engele, M., Sieling, P.A., Barnes, P.F., Rollinghoff, M., Bolcskei, P.L., Wagner, M., Akira, S., Norgard, M.V., Belisle, J.T., Godowski, P.J., Bloom, B.R., Modlin, R.L., 2001. Induction of direct anti-microbial activity through mammalian toll-like receptors. Science 291, 1544–1547. Werling, D., Hope, J.C., Chaplin, P., Collins, R.A., Taylor, G., Howard, C.J., 1999. Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells. J. Leukocyte Biol. 66, 50–58. Whelan, A.O., Hope, J.C., Howard, C.J., Clifford, D., Hewinson, R.G., Vordermeier, H.M., 2003. Modulation of the bovine delayed type hypersensitivity responses to defined mycobacterial antigens by a synthetic bacterial lipopeptide. Infection and Immunity., in press. Wiesmuller, K.H., Jung, G., Hess, G., 1989. Novel low-molecularweight synthetic vaccine against foot-and-mouth disease containing a potent B-cell and macrophage activator. Vaccine 7, 29–33.
Maturation of bovine dendritic cells by lipopeptides Jayne C. Hopea,*, Adam O. Whelanb, R.G. Hewinsonb, Martin Vordermeierb, Chris J. Howarda a
Institute for Animal Health, Compton, Newbury, Berkshire, UK b Veterinary Laboratories Agency, Addlestone, Weybridge, UK
Received 26 August 2002; received in revised form 16 April 2003; accepted 16 April 2003
Abstract The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge. The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells. We report here that bovine DC are also induced to mature following lipopeptide stimulation. Exposure of DC to the model lipopeptide Pam3CSK4 was associated with increased expression of MHC, costimulatory molecules, and enhanced secretion of IL-12 and TNFa. Lipopeptide-matured DC were superior in their ability to induce T cell activation and IFNg secretion. In contrast, exposure of MF to lipopeptides induced down-regulation of MHC expression and much lower increases in IL-12 secretion. A lipopeptide derived from the sequence of a relevant mycobacterial lipoprotein, MPB83, also influenced bovine DC by stimulating increases in IL-12 and TNFa secretion. These different changes in bovine DC and MF may have important implications for immune responses induced following bacterial infection with uptake of microbes by DC resulting in potentiation of their immunostimulatory capacity and uptake by MF having a much less marked effect on immune responses. # 2003 Elsevier B.V. All rights reserved. Keywords: Dendritic cells; Macrophages; Mycobacterium bovis; Lipopeptides
1. Introduction Dendritic cells are unique in their capacity to initiate immune responses. The effects of microbial products on these cells and the effect that these have on DC function have been studied extensively in human and mouse systems. Stimulation or infection of DC with microbes and their products is associated Abbreviations: DC, dendritic cells; TNF, tumour necrosis factor; MF, macrophages; TLR, toll-like receptor; IL, interleukin * Corresponding author. Tel.: þ44-1635-578411; fax: þ44-1635-577263. E-mail address: [email protected] (J.C. Hope).
with maturation of the DC such that their capacity to induce T cell activation is enhanced. These effects on DC are likely to lead to the induction of more efficient immune responses following microbial challenge. For mycobacteria, the lipoproteins expressed by the bacteria are thought to be largely responsible for the maturation events observed in DC (Brightbill et al., 1999; Hertz et al., 2001; Thoma-Uszynski et al., 2001). Thus, human DC infected with M. tuberculosis or M. bovis BCG show enhanced expression of MHC and costimulatory molecules, and also secrete enhanced levels of IL-12 and TNFa (Henderson et al., 1997; Demangel et al., 1999; Kim et al., 1999). This can be mimicked in vitro by the addition
0165-2427/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-2427(03)00104-1
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of the 19 kDa lipoprotein isolated from M. tuberculosis and also by synthetic lipopeptides that are similar in size and hydrophobicity to microbial lipoproteins (Hertz et al., 2001; Thoma-Uszynski et al., 2000). These effects on human DC have been attributed to events downstream of stimulation of toll-like receptor 2 (TLR-2; Hertz et al., 2001; Thoma-Uszynski et al., 2000). Other bacterial products can activate antigen presenting cells via other TLR, for example CpG motifs act via TLR-9 and LPS via TLR-4. The TLR family of receptors are evolutionary conserved receptors present most abundantly on antigen presenting cells in mammals, and are an important element of the innate response to infection (Medzhitov et al., 1997; Krutzik et al., 2001). Stimulation of TLR by microbial products is likely to be an important mechanism by which cells of the innate immune system can recognise and be activated by microbial products, leading to the initiation of an adaptive immune response. The effects of microbial products on macrophages (MF) have also been described and linked to TLR expression. One group has shown that these cells respond to lipopeptides and live M. tuberculosis by down-regulating MHC molecules, a mechanism which may allow immune evasion (Noss et al., 2000, 2001). As MF are considered the major host cell for mycobacteria in vivo their responses may also be pivotal in determining whether the outcome of infection is disease or immunity. Infection of cattle with M. bovis causes bovine tuberculosis, a problem of major concern which is becoming more common in UK cattle herds. Understanding the immune response that occurs following M. bovis infection in cattle will provide important information that might aid better vaccine design or the development of more effective control strategies. The central role of MF in mycobacterial infections is well established. Infection of bovine DC with M. bovis BCG has been reported and these cells are capable of inducing vigorous antigen specific T cell responses in vitro (Hope et al., 2000). The effects of infection with M. bovis or M. bovis BCG on DC maturation are similar to those reported for human DC infected with mycobacteria (Thom and Hope, unpublished data). In this study we have assessed the effect of lipopeptides on bovine DC and MF and we describe changes in a number of parameters that are important for immune regulation. This is the first report directly
comparing the effects of microbial lipopeptides on the phenotype and function of DC and MF derived from the same source and also in a relevant natural target species. We report that lipopeptides induce both functional and phenotypic maturation of DC and also affect MHC expression by MF.
2. Materials and methods 2.1. Lipopeptides The lipopeptide Pam3CSK4, where the Pam3C is represented by a palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl) residue, was obtained from EMC Microcollections, Tubingen, Germany. A lipopeptide based on the N-terminus of M. bovis MPB83 (Pam3CSS) was synthesised by solid phase F-moc chemistry. Initially, a di-amino acid peptide S–S was synthesised on a Model 432 peptide synthesiser (PE-Applied Biosystems, Warrington, UK). Pam3C (Bachem, St. Helens, UK) was dissolved in dichloromethane and then coupled to the amide group of the S–S resin attached peptide in the presence of 2-(1H-benzotriazyl-1-yl)1,1,3,3-tetramethyluranium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA) for 14 h at 20 8C (reagents supplied by PE-Applied Biosystems). The molar ratios of the S–S peptide, Pam3C, HBTU and DIEA in the coupling reaction were 1:3:2.7:3, respectively. A further unmodified control peptide CSS was synthesised on a Model 432 peptide synthesiser. Cleavage of peptides from the resin was performed by TFA hydrolysis. The peptide CSS was recovered from the hydrolysis solution by precipitation in methyl-tert-butyl ether. The recovered peptide was re-suspended in water and lyophilised two consecutive times. Due to the lipophillic nature of the peptide Pam3CSS, the peptide was recovered from the hydrolysis solution by lyophilisation and then resuspended in water and lyophilised further two times. The identity of peptides was confirmed by electrospray mass spectrometry and purity confirmed to be >80% by reverse phase HPLC. The MPB83 lipopeptide Pam3CSS and control peptide CSS are very different in molecular weight (1048.7 and 294.3, respectively). Thus these were tested at equimolar concentrations to ensure accurate comparison of their immunomodulatory properties.
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For comparative purposes with the synthetic lipopeptide Pam3CSK4, 50 mM of Pam3CSS equates to a concentration of 55 mg/ml. All peptides were diluted for use in PBS containing 0.1% Tween and aliquots were stored at 20 8C. Immediately prior to use the peptides were thawed and briefly sonicated to create a uniform suspension. 2.2. Generation of bovine blood derived-dendritic cells and MF Bovine monocytes were cultured with GMCSF and IL-4 to derive DC by a slight modification of the method described previously (Werling et al., 1999). Peripheral blood mononuclear cells (PBMC) were derived by centrifugation over Histopaque (Sigma), and were incubated with anti-human CD14 labelled super-paramagnetic particles (Miltenyi-Biotech, Bergisch Gladbach, Germany), and labelled cells were isolated from a Midimacs column (Miltenyi-Biotech) according to the manufacturers instructions. The purity of the cells was evaluated by flow cytometry and shown in each case to be >98%. Cell viability was >95%. Cells were adjusted to 8 105 ml1 in RPMI-1640 medium containing Glutamax-1 (Life Technologies, Paisley, UK), 10% heat inactivated FCS, 5 105 M 2-ME, 50 mg/ml gentamycin (tissue culture medium; TCM), 200 U/ml COS-7 cell derived bovine rIL-4 (Hope et al., 2000) and 0.2 U/ml bovine rGMCSF (units based on induction of 1/2 maximal proliferation in bone marrow precursor cells) and 3 ml of this suspension was added per well of sixwell plates. After 3 days of culture, the peptides were added to give the indicated final concentrations and the DC were cultured for an additional 2 days. On day 5 of culture the DC were harvested and counted. MF were CD14þ cells isolated as described above and cultured for 3 days in IMDM (Life Technologies) containing 10% FCS, 5 105 M 2-Me and 50 mg/ml gentamycin. Peptides were added on day 3 of culture and the MF cultured for a further 2 days prior to analysis. 2.3. Flow cytometry The mouse anti-bovine mAb used were CC158 (anti-MHC class II; IgG2a), IL-A88 (anti-MHC
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class I; IgG2a), CC14 (anti-CD1b; IgG1), IL-A156, IL-A159, IL-A190 (anti-CD40, CD80 and CD86, respectively, all IgG1; provided by Dr. N. MacHugh, Centre for Tropical Medicine, Roslin, UK). Control mAb used within the study were AV20 (mouse IgG1) and AV37 (mouse IgG2a), that are directed against chicken bursal B cells, chicken CD4þ cells and chicken spleen cell subset, respectively, all provided by Dr. T.F. Davison, IAH. DC or MF were incubated with primary mAb at pre-determined optimal concentrations for 10 min, then washed extensively. Bound mAb was detected with FITC labelled anti-mouse IgG (Southern Biotechnologies, Birmingham, AL, USA). The cells were analysed on a FACSCalibur (Becton Dickinson) and immunofluorescent staining was analysed using Win-MDI software. 10,000 gated cells were analysed for each parameter. 2.4. Proliferation assays and measurement of IFNg Allogeneic PBMC (105 per well) were incubated in triplicate with 1 104 irradiated MF or DC (20 Gy from a 137 Cssource) in a total volume of 200 ml TCM. Cultures were incubated for 5 days at 37 8C. 37mBq [3 H]-thymidine (3 HTdR; DuPont, Stevenage, UK) was added for the final 18 h of culture. Results are expressed as mean S:D: of triplicate wells. In some experiments supernatants were harvested from parallel cultures on day 4 and IFNg was measured by ELISA as previously described (15). Results are expressed as pg/ml. 2.5. TNFa and IL-12 ELISA Levels of TNFa and IL-12 in culture supernatants derived from MF or DC were assessed by ELISA. Antibodies to TNFa and IL-12 were generated by immunisation of mice with recombinant bovine TNFa or IL-12, and hybridomas were generated according to a standard method (Kwong et al., 2002). Pairs of mAb were tested for suitability in ELISA and both recombinant and natural TNFa or IL-12 were shown to be detected in an ELISA using mAbs CC327 and biotinylated-CC328 for TNFa (unpublished data), or mAbs CC301 and biotinylated-CC326 for IL-12 (Hope et al., 2002). The IL-12 ELISA was performed using the protocol as described by Kwong et al. (2002).
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For the TNFa ELISA, black microplates (Porvair, Shepperton, UK) were coated with 2 mg/ml CC327 and incubated overnight at room temperature. The plates were washed in washing buffer and blocking buffer was added for 1 h. Following a further washing step, samples were added for 1 h. Dilutions of the standard preparation of COS-7 cell derived rbo TNFa (in blocking buffer) were also added to the plates for 1 h. Following washing CC328-biotin (1 mg/ml in blocking buffer) was added for 1 h, followed by washing and addition of SA-HRP for 45 min. Following the final washing step the substrate
(Super Signal ELISA femto maximum sensitivity substrate; Pierce) was added for 1 min and relative light units (RLU) were read on an Anthos LUCY 1.0 luminometer (Anthos Labtec, Salzburg, Austria). Units per millilitre were calculated in comparison with the standard. 2.6. Statistical analysis Statistical analyses were performed using a paired Student’s t-test. P-values of less than 0.05 were considered significant.
Fig. 1. Phenotypic analysis of DC exposed to lipopeptides. Dendritic cells were exposed to Pam3CSK4 at the indicated concentrations for 48 h. Expression of the indicated surface molecules was assessed by flow cytometry. The results are expressed as mean fluorescence intensity (MFI) of cells within the live cell gate. One representative experiment of four is shown.
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3. Results 3.1. Stimulation of bovine DC with lipopeptides induced increases in expression of CD40, CD80 and CD1 On day 3 of DC culture, the lipopeptide Pam3CSK4 was added. After a further 2 days of culture the DC were harvested and analysed by flow cytometry for the expression of MHC class I, MHC class II, CD40, CD80, CD86 and CD1b. Following exposure of DC to Pam3CSK4 we observed a dose-dependent increase in the level of CD40, CD80, CD86 and CD1b (Fig. 1). More variable effects on expression of MHC class I and MHC class II were observed. In some experiments expression was unaffected by treatment, whereas in others both MHC I and MHC II were increased following exposure of DC to lipopeptide (Fig. 1). No effects on binding of control mAb were observed under any conditions (data not shown). 3.2. IL-12 and TNFa secretion by DC following culture with lipopeptides Exposure of DC to Pam3CSK4 was associated with a dose-dependent increase in IL-12 secretion with effects observed after stimulation with as low as 0.1 mg/ml of lipopeptide (Fig. 2a). In addition, treatment of DC with Pam3CSK4 led to a dose-dependent increase in TNFa secretion (Fig. 2b). 3.3. Increased allogeneic stimulation of T cells following culture of DC with lipopeptides In a further set of experiments, the capacity of DC to induce proliferation and to produce IFNg after stimulation with allogeneic PBMC was assessed. Following culture of DC with Pam3CSK4 there was a small but significant increase in the capacity of DC to stimulate allogeneic PBMC (P < 0:05; data not shown). In addition, stimulation of allogeneic PBMC with DC cultured in the presence of Pam3CSK4 (¼0.1 mg/ml) significantly increased the secretion of IFNg (P < 0:05; Fig. 2c). 3.4. Effects of lipopeptides on bovine MF Monocytes were cultured for 3 days then exposed to Pam3CSK4 for an additional 48 h. The effects on
Fig. 2. Lipopeptide stimulated DC secrete IL-12 and TNFa and induce increased IFNg synthesis by allogeneic PBMC. Dendritic cells were exposed to Pam3CSK4 at the indicated concentrations for 48 h. Supernatants were taken and assayed for IL-12 (a) and TNFa (b) by ELISA. The results are shown as biological units per millilitre. For MLR 104 stimulated APC were added to 105 allogeneic PBMC (c). Supernatants were derived after 4 days of culture and assayed by ELISA for the presence of IFNg. Results are expressed as pg/ml. One representative experiment of four is shown.
CD40, CD80, CD86 and CD1 expression on MF were noted only at the highest concentration of lipopeptide tested (Fig. 3). These were paralleled by increases in non-specific binding of control mAb (data not shown). However, addition of lipopeptide to cultures of MF decreased the expression of MHC class I and MHC class II in a dose-dependent manner (Fig. 3).
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Fig. 3. Effect of lipopeptide exposure on bovine MF surface molecule expression. MF were exposed to Pam3CSK4 at the indicated concentrations for 48 h. Expression of the indicated surface molecules was assessed by flow cytometry. The results are expressed as MFI of cells within the live cell gate. One representative experiment of three is shown.
The capacity of MF to induce allogeneic MLR proliferation (data not shown) and IFNg responses (Fig. 4c) was not affected when the cells were exposed to Pam3CSK4. In addition, much lower levels of IL-12 and TNF secretion were induced upon lipopeptide stimulation of compared to DC. 3.5. Effects of M. bovis derived lipopeptide on bovine DC To assess whether lipopeptides derived from the sequences of mycobacterial lipoproteins could also elicit changes in bovine DC and MF, a lipopeptide was synthesised based on the N-terminus of MPB83, a major secreted lipoprotein of M. bovis.
Amino-terminal sequencing of a native form of MPB83 from a M. bovis culture supernatant previously identified the loss of the first three amino acids (C–S–S) from the mature form of the protein (Whelan et al., 2003). Since the site of lipid modification of MPB83 is predicted to be at the amino-terminal Cys residue of the mature protein (Hewinson et al., 1996), the synthesis of the peptide Pam3CSS represented a biological relevant mycobacterial lipopeptide. The lipopeptide or non-lipoylated control peptides were added to DC and IL-12 secretion was assessed after 48 h of culture. The MPB83-derived lipopeptide (Fig. 5, black bars) induced dose-dependent increases in IL-12 (Fig. 5a) and TNFa (Fig. 5b) secretion from DC that were significantly different from those induced
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Fig. 4. Effects of lipopeptide stimulation on MF cytokine secretion and MLR inducing capacity. MF were exposed to Pam3CSK4 at the indicated concentrations for 48 h. Supernatants were taken and assayed for IL-12 (a) and TNFa (b) by ELISA. The results are shown as biological units per milliliter. For MLR 104 stimulated APC were added to 105 allogeneic PBMC (c). Supernatants were derived after 4 days of culture and assayed by ELISA for the presence of IFNg. Results are expressed as pg/ml. One representative experiment of four is shown.
by the control peptide (P < 0:05; Fig. 5, open bars). In contrast stimulation of MF with the MPB83-derived lipopeptide did not induce IL-12 secretion (data not shown).
4. Discussion DC are unique in their capacity to activate naive lymphocytes, resulting in the initiation of immune
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Fig. 5. Effect of M. bovis lipopeptide on cytokine expression by bovine DC. Dendritic cells were exposed to the M. bovis MPB83 lipopeptide Pam3CSS at the indicated concentrations for 48 h. Supernatants were taken and assayed for IL-12 (a) and TNFa (b) by ELISA. The results are shown as biological units per milliliter. One representative experiment of two.
responses. A number of studies have suggested that microbial products, including lipopeptides and lipoproteins, may act upon DC to enhance their antigen presenting and costimulatory capacity (Hertz et al., 2001; Thoma-Uszynski et al., 2001). Stimulation of DC with mycobacteria increases expression of MHC and costimulatory molecules, as well as enhancing their secretion of immunoregulatory cytokines such as IL-12 (Henderson et al., 1997; Demangel et al., 1999). We have demonstrated that culture of immature bovine DC with synthetic lipopeptides increases cell surface expression of CD40, CD80, CD86 and CD1, resulting in cells with increased T cell-stimulatory capacity when compared to cells cultured without lipopeptide. Functionally, the lipopeptide-matured
DC have enhanced T cell-stimulatory activity in allogeneic MLR and can enhance IFNg synthesis by allogeneic PBMC. In addition, lipopeptide stimulated DC expressed increased levels of IL-12 and TNFa, consistent with maturation. Moreover, we have shown that a lipopeptide based on the M. bovis secreted protein MPB83 also induces these maturation events, providing evidence that the DC maturation that is observed upon infection with M. bovis (Hope, unpublished observations) is, at least in part, induced by lipopeptides secreted from the mycobacterium. Increases in secretion of IL-12 and TNFa, alongside an enhanced capacity to stimulate T cells present within PBMC may have important implications for the induction of effective immune responses against M. bovis.
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Previous studies have demonstrated that stimulation with LPS or with live bacteria induces DC maturation. Stimulation of immature DC with LPS from Escherichia coli (Sallusto and Lanzavecchia, 1994) induced increases in a number of cell surface markers, including MHC class II, CD40, CD80, CD54, and CD58, whereas expression of CD14, CD32, and endocytic activity was reduced. In addition, infection of human DC with live bacillus Calmette-Gue´ rin (Kim et al., 1999), M. tuberculosis (Henderson et al., 1997), or Listeria monocytogenes (Kolb-Maurer et al., 2000), results in an increase in MHC and costimulatory molecule expression and enhanced T cell-stimulatory activity. Similar results were obtained with murine DC exposed to M. tuberculosis. Following infection of murine DC, expression of MHC and costimulatory molecules was upregulated and these DC showed enhanced ability to induce T cell responses (Demangel et al., 1999). Studies by Hertz et al., demonstrated that the 19 kDa lipoprotein from M. tuberculosis, as well as synthetic lipopeptides, induced DC maturation. The resulting mature DC had increased cell surface expression of MHC class II, CD80, CD83, CD86, CD54, and CD58, suggesting that the lipopeptide alone is sufficient to induce maturation events (Hertz et al., 2001). Lipopeptide-matured DC were also more potent than immature DC in stimulating T cells in a MLR (Hertz et al., 2001). Together, these findings provide a mechanism by which cells of the innate immune system can recognise and be activated by microbial products, leading to the initiation of an adaptive immune response. The study described herein illustrates that bovine DC respond to lipopeptides in a similar way, by upregulating expression of MHC and costimulatory molecules, including cytokines. A lipopeptide from the M. bovis MPB83 protein also increased the secretion of immunologically relevant cytokines by bovine DC. These effects are likely to reflect those observed following infection of DC by mycobacteria. In preliminary studies we have shown that infection of DC with virulent M. bovis or M. bovis BCG does indeed enhance expression of IL-12, TNFa and surface molecules including CD40 and CD80 (Hope and Thom, unpublished results). In addition to the modulation of immune responses by the lipid moiety, we have data that allows us to hypothesise that the length of the amino acid sequence attached to the lipopeptide, or its hydrophobicity relative to the lipid
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moiety, might be important for the induction of DC maturation. Thus, the longer MPB83-derived lipopeptide Pam3CSSTKPVSQDTSPKPA did not induce IL-12 secretion or phenotypic changes in DC (data not shown), whereas the shorter and more hydrophobic lipopeptide Pam3CSS exhibited significant biological effects. The production of IL-12 by DC infected with microbial pathogens is likely to have important implications for the outcome of infection. IL-12 is known to one of the most important mediators determining Th1 bias of the immune response (Macatonia et al., 1993). Th1 biased immunity is regarded as being central to effective immune responses against a number of bacterial pathogens. In this study, lipopeptide-matured DC showed enhanced capacity to stimulate IFNg secretion from allogeneic PBMC indicating that there may be more effective stimulation of Th1 responses. The increased secretion of IL-12 by lipopeptidematured DC may also stimulate NK cells which may contribute to the secretion of IFNg. Upregulation of costimulatory molecules and MHC may also contribute to the induction of more effective immune cell activation. Taken together the result of lipopeptide exposure of DC is likely to be initiation of Th1 cell activation. In addition to effects on DC, synthetic lipopeptides derived from the N-terminus of bacterial lipoproteins of the kind we have used in this study have been described as potent activators of murine and human MF and monocytes and polyclonal stimulators of murine B cells (Bessler et al., 1997a,b). They are also efficient immunoadjuvants in parenteral, oral and nasal immunisation either in combination with, or after covalent linkage to an antigen. Enhanced antibody-mediated immune responses and enhanced cellular immune responses in vivo have been reported after vaccination with lipopeptide–antigen conjugates, including the induction of MHC class I restricted CTL (Bessler et al., 1997a,b; Wiesmuller et al., 1989; Deres et al., 1989). The present paper is, to our knowledge, the first study describing biological activities of synthetic bacterial lipopeptides on bovine leukocytes and provides important comparisons of the effects of lipopeptides on different types of antigen presenting cells. Interestingly, in contrast to studies in other species like mice and humans that demonstrated an effect of such lipopeptides on MF, e.g. TNF-a
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production (Hauschildt et al., 1990; Hoffmann et al., 1988), we could not demonstrate significant lipopeptide induced cytokine production (IL-12 or TNF-a) by bovine MF. The most notable effect on bovine MF observed in the present investigation was the downregulation of MHC expression following lipopeptide exposure. Other studies have reported similar effects on MHC expression in murine and human MF following exposure to lipopeptides or to mycobacteria (Noss et al., 2000; Hauschildt et al., 1990). The downregulation of MHC class II may be a mechanism by which mycobacteria resident within MF evade detection by reactive T lymphocytes thus enabling survival and perpetuation of the infection. Extending the in vitro data presented in the present study to in vivo responses, we recently demonstrated that co-injection into cattle of lipopeptide with a skin test antigen significantly enhanced the skin test response. In 4/7 M. bovis infected calves tested Pam3CSK4 enhanced the skin test reactivity against the test antigen ESAT-6 (Whelan et al., 2003). These results suggest that lipopeptides may serve as effective vaccine adjuvants in cattle as well as other species. The mechanism by which microbe-induced DC maturation occurs is mediated by toll-like receptors (TLR; Krutzik et al., 2001). TLRs are a family of at least 10 transmembrane proteins that are evolutionarily conserved in many species (Medzhitov et al., 1997). The result of signalling through TLR is induction of the NFkB signalling pathway and the production of cytokines (Brightbill et al., 1999). Lipopeptides and lipoproteins activate cells of the immune system in a TLR-2 dependent manner (Hertz et al., 2001; Thoma-Uszynski et al., 2000; Noss et al., 2001). We have recently shown that bovine DC express RNA for TLR-2 (Hope and Werling, unpublished data) suggesting that lipopeptides could signal through this pathway in the bovine system. This study illustrates that in M. bovis the lipopeptides are likely to be important in inducing events following infection with pathogenic bacteria and provides data regarding the effects of microbes on DC and MF that may be relevant to the pathogenesis of disease. References Bessler, W.G., Heinevetter, L., Wiesmuller, K.H., Jung, G., Baier, W., Huber, M., Lorenz, A.R., Esche, U.V., Mittenbuhler,
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