This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jebbari, H. Articles by Knight, S. C. Search for Related Content PubMed PubMed Citation Articles by Jebbari, H. Articles by Knight, S. C.

Leishmania major Promastigotes Inhibit Dendritic Cell Motility In Vitro

Antigen Presentation Research Group, Department of Immunology,¹ Department of Infectious Diseases and Tropical Medicine, Imperial College School of Medicine, Northwick Park Hospital, Harrow, London HA1 3UJ, United Kingdom²

Received 25 April 2001/ Returned for modification 4 June 2001/ Accepted 12 November 2001

	ABSTRACT

Top Abstract Introduction Spleen dc display high... Leishmania inhibit DC motility. Inhibition of dc motility... References

 
Using an in vitro transwell migration assay, we have demonstrated that products secreted by Leishmania major promastigotes inhibit the motility of dendritic cells (DC) by up to 93%. Inhibition was dose dependent and reversible. By inhibiting DC migration in vivo, L. major may therefore subvert DC from their potentially protective role during leishmaniasis.

	INTRODUCTION

Top Abstract Introduction Spleen dc display high... Leishmania inhibit DC motility. Inhibition of dc motility... References

 
Resistance against Leishmania follows from the activation of Th1 CD4⁺ T-cell-mediated immunity (reviewed in reference 6). The production of interleukin 12 (IL-12), a cytokine that promotes the development of a Th1 response, is detectable in the early stages of experimental Leishmania major infection (5, 6, 8). Dendritic cells (DC) produce IL-12 following stimulation with L. major in vitro (8, 22) and can be found bearing parasites in lymph nodes draining the site of infection (11); these DC are able to stimulate Leishmania-specific T cells (8, 12). The initiation of a primary immune response requires the interaction of antigen-presenting DC with recirculating naive T cells in secondary lymphoid tissue. Therefore, movement of DC from the skin to the draining lymph nodes is a critical step in the initiation of a response to cutaneous Leishmania infection. Isolated DC are characteristically motile cells (7), but here we report that L. major is able to inhibit DC motility in vitro and suggest that this may be a further means by which the parasite can subvert the development of a protective immune response.

	Spleen DC display high spontaneous motility.

Top Abstract Introduction Spleen dc display high... Leishmania inhibit DC motility. Inhibition of dc motility... References

 
Partially enriched DC from 6- to 8-week-old BALB/c mice were isolated after overnight culture of spleen cells in complete tissue culture medium: Dutch modified RPMI 1640 (Gibco, Paisley, United Kingdom) supplemented with 10% (vol/vol) fetal calf serum (Gibco, Grand Island, N.Y.), 2 mM L-glutamine, 100 U of penicillin/ml, and 100 µg of streptomycin/ml. Metrizamide (13.7%) was used (Nygaard, Oslo, Norway) as previously described (8) to obtain low-density cells (LDC). Spontaneous migration of these cells (10⁵/100 µl) from the upper to the lower chamber of 3-µm-pore-size Transwells (Costar, Cambridge, Mass.) was assayed after incubation for 1 h at 37°C in 5% CO₂. Migrating cells were quantitated by one of two methods: (i) cytospins were prepared from the contents of each lower chamber and were fixed and stained with modified Wright Giemsa (Sigma, Dorset, United Kingdom), and cells were counted microscopically, or (ii) cellular ATP was assayed in the lower chamber with a ViaLight HS ATP kit (LumiTech, Workingham, United Kingdom). Luminescence from the luciferase-catalyzed reaction between ATP and luciferin was measured with a Turner Designs luminometer (TD20/20) set up for a 3-s delay and a 10-s integration time. A linear relationship exists between ATP content and cell number; results obtained by this method are expressed as relative luminescence units (RLU).

When complete tissue culture medium was placed in the lower chamber, approximately 1.2% of the input cells migrated during the culture period. Immunomagnetic separation of the input cells with a Minimax column (labeling with anti-CD11c microbeads; Miltenyi, Bisley, United Kingdom) and FACScan analysis (labeling with fluorescein isothiocyanate-labeled anti-CD11c antibodies; Pharmingen, Oxford, United Kingdom) confirmed that CD11c⁺ DC but not contaminating CD11c^- cells displayed spontaneous migration (Fig. 1).

View larger version (15K): [in this window] [in a new window]   FIG. 1. (A) Spleen LDC were separated immunomagnetically into CD11c+ and CD11c- (non-DC) fractions, and purity was assessed by flow cytometry. (B) The cells displaying spontaneous motility were found in the CD11c+ fraction.

	Leishmania inhibit DC motility.

Top Abstract Introduction Spleen dc display high... Leishmania inhibit DC motility. Inhibition of dc motility... References

View larger version (35K): [in this window] [in a new window]   FIG. 2. The inhibitory effects of Leishmania parasites on DC motility when the parasites were either present alive (PCM) or dead (F/Th-PCM) or were removed from their conditioned medium (F-PCM) (A), when F-PCM was used neat or, when diluted (B), or when DC were placed in new medium to show if the inhibition of motility was reversible (C).

	Inhibition of DC motility by PCM is dose dependent and reversible.

Top Abstract Introduction Spleen dc display high... Leishmania inhibit DC motility. Inhibition of dc motility... References

PCM-induced inhibition of DC motility is reversible. Transwells containing DC were incubated for 1 h in either medium alone or PCM and then were transferred to new wells containing fresh medium. Following a second 1-h incubation, the Transwells were again moved to new wells containing fresh medium. After all incubations were complete, medium from the bottom chambers was collected and the number of migrated cells was determined. PCM-mediated inhibition of DC motility (66% after 1 h) was reversible when DC had been removed from the PCM for 1 and 2 h (Fig. 2C).

DC are characteristically motile cells in vitro (2, 7, 10, 18, 19, 20), and this work demonstrates that products of L. major promastigotes inhibit this movement of DC in a dose-dependent and reversible manner. This effect was not due to toxicity, since DC retained their viability (trypan blue exclusion) and recovered their motility after removal from the parasite-conditioned medium. In addition, parasites added to fresh medium inhibited DC migration, arguing against an effect of toxic metabolites accumulating in the parasite-conditioned medium. Whether the reduced DC motility observed was due to parasite-induced changes in DC adhesion molecules has still to be investigated; however, previous work (8) has shown that coculture of DC with promastigotes does not affect the general aspects of DC activation.

Migration, from the skin to the regional lymph nodes, of DC bearing parasite antigens is likely to be a pivotal step in the generation of protective immunity to Leishmania (11). While it may be possible that perturbations could occur within DC subsets, leading to exacerbation of disease, the literature presently points towards DC migration having a protective role in leishmaniasis (1, 3, 9, 11, 12, 13, 21). Thus, inhibition of this migration could be a further means by which the parasite can subvert the development of a protective host response. The movement of DC in vivo is presently not fully understood but probably involves cytokine- or antigen-mediated regulation of DC chemokine receptor expression. This may enable the DC to move in response to inflammatory or constitutive chemokines at different stages of their life history. Presently the relationship between these events and the spontaneous migration of DC we have measured in vitro is an open question. Ultimately, in vivo infection experiments will be required to test the significance of our observations.

Leishmania inhibits the motility and chemotaxis of various cells, including monocytes (4), polymorphonuclear neutrophils (16), and phagocytes (17). Agonist and antagonist effects of Leishmania upon cell migration and chemotaxis have been reported; this could be accounted for by the use of different strains of parasite and life cycle stages and dissimilar preparations of the parasite (4, 16, 17). The Leishmania component responsible for inhibition of DC motility is currently unidentified; however, preliminary findings identified the active part of PCM to be <10 kDa (data not shown). Thus, an excreted/secreted component, such as the major surface antigen lipophosphoglycan (LPG), is one possibility (4, 16, 17), since LPG varies in size between 6 and 100 kDa (14, 15), depending upon various factors of culture technique and parasite life cycle stage.

These observations have significance for vaccine development and immunotherapy. In particular, the reversible nature of the block in DC movement may mean that therapeutic agents which can overcome the effect of the parasite and stimulate movement of antigen-bearing DC into the lymph nodes may be of value in infected individuals.

	ACKNOWLEDGMENTS

Top Abstract Introduction Spleen dc display high... Leishmania inhibit DC motility. Inhibition of dc motility... References

 
Financial support for this work was provided by the Wellcome Trust.

We are grateful to the Pfizer research group at Northwick Park Hospital, Harrow, for the use of their luminometer.

	FOOTNOTES

 
* Corresponding author. Mailing address: Mycobacterium Reference Unit, Dulwich Public Health Laboratory, Dulwich Hospital, East Dulwich Grove, London SE22 8QF, United Kingdom. Phone: 020 8693 1312. Fax: 020 7436 6477. E-mail: hjebbari{at}yahoo.co.uk.

Editor: J. M. Mansfield

	REFERENCES

Top Abstract Introduction Spleen dc display high... Leishmania inhibit DC motility. Inhibition of dc motility... References

 

1.	Arnoldi, J., and H. Moll. 1998. Langerhans cell migration in murine cutaneous leishmaniasis: regulation by tumour necrosis factor alpha, interleukin-1 beta, and macrophage inflammatory protein-1 alpha. Dev. Immunol. 6:3-11.[Medline]
2.	Barratt-Boyes, S. M., M. I. Zimmer, L. A. Harshyne, E. M. Meyer, S. C. Watkins, S. Capuano, M. Murphey-Corb, L. D. Falo, and A. D. Donnenberg. 2000. Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines. J. Immunol. 164:2487-2495.[Abstract/Free Full Text]
3.	Flohe, S. B., C. Bauer, S. Flohe, and H. Moll. 1998. Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major. Eur. J. Immunol. 28:3800-3811.[CrossRef][Medline]
4.	Frankenburg, S., A. Gross, and V. Leibovici. 1992. Leishmania major and Leishmania donovani: effect of LPG-containing and LPG-deficient strains on monocyte chemotaxis and chemiluminescence. Exp. Parasitol. 75:442-448.[CrossRef][Medline]
5.	Guler, M. L., N. G. Jacobson, U. Gubler, and K. M. Murphy. 1997. T cell genetic background determines maintenance of IL-12 signaling-effects on BALB/c and B10.D2 T helper cell type 1 phenotype development. J. Immunol. 159:1767-1774.[Abstract]
6.	Jebbari, H., and R. N. Davidson. 1998. Recent advances in leishmaniasis. Curr. Opin. Infect. Dis. 11:535-539.
7.	Knight, S. C., and A. J. Stagg. 1993. Antigen-presenting cell types. Curr. Opin. Immunol. 5:374-382.[CrossRef][Medline]
8.	Konecny, P., A. J. Stagg, H. Jebbari, N. English, R. N. Davidson, and S. C. Knight. 1999. Murine dendritic cells internalise Leishmania major promastigotes, produce IL-12 and stimulate primary T cell proliferation in vitro. Eur. J. Immunol. 29:1803-1811.[CrossRef][Medline]
9.	Kremer, I., M. P. Gould, K. D. Cooper, and F. P. Heinzel. 2001. Pre-treatment with recombinant Flt3 ligand partially protects against cutaneous leishmaniasis in susceptible BALB/c mice. Infect. Immun. 69:673-680.[Abstract/Free Full Text]
10.	Maestroni, G. J. 2000. Dendritic cell migration controlled by alpha 1b-adrenergic receptors. J. Immunol. 165:6743-6747.[Abstract/Free Full Text]
11.	Moll, H., H. Fuchs, C. Blank, and M. Rollinhoff. 1993. Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells. Eur. J. Immunol. 23:1595-1601.[Medline]
12.	Moll, H., S. Flohe, and M. Rollinghoff. 1995. Dendritic cells in Leishmania major-immune mice harbor persistent parasites and mediate an antigen- specific T-cell immune response. Eur. J. Immunol. 25:693-699.[Medline]
13.	Moll, H. 1997. The role of chemokines and accessory cells in the immunoregulation of cutaneous leishmaniasis. Berhing Inst. Mitt. Mar:73-78.
14.	Moody, S. F., E. Handman, and A. Bacic. 1991. Structure and antigenicity of the lipophosphoglycan of Leishmania major amastigotes. Glycobiol. 1:419-424.[Abstract/Free Full Text]
15.	Moody, S. F., E. Handman, M. J. McConville, and A. Bacic. 1993. The structure of Leishmania major amastigote lipophosphoglycan. J. Biol. Chem. 268:18457-18466.[Abstract/Free Full Text]
16.	Panaro, M. A., M. Panunzio, E. Jirillo, A. Marangi, and O. Brandonisio. 1995. Parasite escape mechanisms: the role of Leishmania lipophosphoglycan on human phagocyte functions. A review. Immunopharmacol. Immunotoxicol. 17:595-605.
17.	Panaro, M. A., V. Puccini, S. M., Faliero, R. Marzio, A. Marangi, S. Lisi, and O. Brandonisio. 1996. Leishmania donovani lipophosphoglycan (LPG) inhibits respiratory burst and chemotaxis of dog phagocytes. Microbiologica 19:107-112.[Medline]
18.	Price, A. A., M. Cumberbatch, I. Kimber, and A. Ager. 1997. Alpha 6 integrins are required for Langerhans cell migration from the epidermis. J. Exp. Med. 186:1725-1735.[Abstract/Free Full Text]
19.	Richters, C. D., E. A. Reits, A. M. Van Pelt, M. J. Hoekstra, J. Van Baare, J. S. Du Pont, and E. W. Kamperdijk. 1996. Effect of low dose UVB irradiation on the migratory properties and functional capacities of human skin dendritic cells. Clin. Exp. Immunol. 104:191-197.[CrossRef][Medline]
20.	Roberts, G. M., and I. Laffafian. 1996. Human cord blood-derived large myeloid cells: spontaneous shape change and chemotactic movement. Br. J. Biomed. Sci. 53:96-100.[Medline]
21.	Sato, N., S. K. Ahuja, M. Quinones, V. Kostecki, R. L. Reddick, P. C. Melby, W. A. Kuziel, and S. S. Ahuja. 2000. CC chemokine receptor (CCR)2 is required for Langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells: absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, B cell outgrowth, and sustained neutrophilic inflammation. J. Exp. Med. 192:205-218.
22.	Von Stebut, E., Y. Belkaid, T. Jakob, D. L. Sacks, and M. C. Udey. 1998. Uptake of Leishmania major amastigotes results in activation and Interleukin-12 release from skin derived dendritic cells: implications for initiation of anti-Leishmania immunity. J. Exp. Med. 188:1547-1552.[Abstract/Free Full Text]

This article has been cited by other articles:

• Colmenares, M., Corbi, A. L., Turco, S. J., Rivas, L. (2004). The Dendritic Cell Receptor DC-SIGN Discriminates among Species and Life Cycle Forms of Leishmania. J. Immunol. 172: 1186-1190 [Abstract] [Full Text]  
• Rotta, G., Edwards, E. W., Sangaletti, S., Bennett, C., Ronzoni, S., Colombo, M. P., Steinman, R. M., Randolph, G. J., Rescigno, M. (2003). Lipopolysaccharide or Whole Bacteria Block the Conversion of Inflammatory Monocytes into Dendritic Cells In Vivo. J. Exp. Med. 198: 1253-1263 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jebbari, H. Articles by Knight, S. C. Search for Related Content PubMed PubMed Citation Articles by Jebbari, H. Articles by Knight, S. C.