The Human Immune Response during Cutaneous ... - Cell Press

Focus 10 Wang, P. et al. (1997) Rnr4p, a novel ribonucleotide reductase small subunit protein. Mol. Cell. Biol. 17, 6114–6121 11 Huang, M. and Elledge, S.J. (1997) Identification of RNR4, encoding a second essential small subunit of RR in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 6105–6113 12 Rubin, H. et al. (1993) Cloning, sequence determination and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum – a target for anti-malarial therapy. Proc. Natl. Acad. Sci. U. S. A. 90, 9280–9284 13 Chakrabarti, D. et al. (1993) Cloning and characterization of the subunit genes of ribonucleotide reductase, a cell cycle regulated enzyme from Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 90, 12020–12024 14 Lye, L.F. et al. (1997) Cloning and functional analysis of the ribonucleotide reductase gene small subunit from hydroxyurearesistant Leishmania mexicana amazonensis. Mol. Biochem. Parasitol. 90, 353–358 15 Hofer, A. et al. (1997) Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei. Proc. Natl. Acad. Sci. U. S. A. 94, 6959–6964 16 Mann, G.J. et al. (1991) Purification and characterisation of recombinant mouse and Herpes simplex virus ribonucleotide reductase R2 subunit. Biochemistry 30, 1939–1947 17 Yang, F. et al. (1997) Characterisation of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit. J. Bacteriol. 179, 6408–6415 18 Cavalier-Smith, T. (1993) Kingdom protozoa and its 18 phyla. Microbiol. Rev. 57, 953–994

19 Wong, J.T. (1996) Protozoan cell cycle control. Biol. Signals 5, 301–308 20 Leete, T.H. and Rubin, H. (1996) Malaria and the cell cycle. Parasitol. Today 12, 442–444 21 Bjorklund, S. et al. (1993) Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase. Proc. Natl. Acad. Sci. U. S. A. 90, 11323–11326 22 Bjorklund, S. et al. (1990) S phase specific expression of mammalian ribonucleotide reductase R1 and reductase R2 subunit messenger RNAs. Biochemistry 29, 5452–5458 23 Nocentini, G. (1996) Ribonucleotide reductase inhibitors – new strategies for cancer chemotherapeutics. Crit. Rev. Oncol. Hematol. 22, 89–126 24 Dutia, B.M. et al. (1986) Specific inhibition of herpes virus ribonucleotide reductase by synthetic peptides. Nature 321, 439–441 25 Luizzi, M. et al. (1994) A potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo. Nature 372, 695–697 26 Fisher A., Laub, P. and Cooperman, B. (1995) NMR structure of an inhibitory R2 C-terminal peptide bound to mouse ribonucleotide reductase R1 subunit. Nat. Struct. Biol. 2, 951–955 27 Flores, M. et al. (1997) Inhibition of Plasmodium falciparum proliferation in vitro using ribozymes. J. Biol. Chem. 272, 16940–16945 28 Barker, R.H. et al. (1996) Inhibition of Plasmodium falciparum malaria using anti-sense oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 93, 514–518 29 Toulme, J. et al. (1997) Control of gene expression in viruses and protozoan parasites by anti-sense oligonucleotides. Parasitology 114, S45–S59

The Human Immune Response during Cutaneous Leishmaniasis: NO Problem M.D. Mossalayi, M. Arock, D. Mazier, P. Vincendeau and I. Vouldoukis During some helminth infections, increased expression of the low-affinity receptor for IgE (CD23/FceRII) by macrophages and/or increased levels of plasma IgE have been seen, but their role in host protection or disease progression remains unclear. Recently, crosslinking of CD23 was shown to promote intracellular killing of Leishmania parasites in human macrophages, a phenomenon involving the production of tumor necrosis factor a and nitric oxide (NO). Based upon various in vitro and in vivo studies of human cutaneous leishmaniasis, Djavad Mossalayi, Michel Arock, Dominique Mazier, Philipe Vincendeau and Ioannis Vouldoukis here propose a model for an immune response that involves CD23– IgE-mediated NO release during protection, as well as during active cutaneous leishmaniasis. The roles of immune cells and their various mediators in healing or progression of leishmaniasis remain unclear. Macrophage-derived cytokines and nitric oxide (NO) kill Leishmania major in murine cells1–3. In human macrophages, leishmanicidal activity was observed in L. major- or L. infantum-infected cells following their in M. Djavad Mossalayi and Michel Arock are at the Hematology Laboratory, Faculty of Pharmacy Paris V, 4 Avenue de l’Observatoire, 75006 Paris, France. Dominique Mazier and Ioannis Vouldoukis are at Inserm U313, Pitié-Salpêtrière Hospital, Paris, France. Phillipe Vincendeau is at the Parasitology Laboratory, Bordeaux 2 University, Bordeaux, France. Tel: +33 1 53 73 97 12, Fax: +33 1 40 46 96 55, e-mail: Djavad.Mossalayi@ umr5540.u-bordeaux2.fr 342

vitro activation by various factors4, including interferon g (IFN-g), or crosslinking of the CD23/FceRII activation antigen5. In an attempt to clarify the role of IgE, CD23 and proinflammatory factors in human Leishmania infection, we compared the in vivo expression of various mediators, as reported by different groups, with their effect on the in vitro leishmanicidal activity of human macrophages. These findings provide a model whereby an immune network that includes T cells and macrophages, as well as B cells, mediates pathogen killing or disease chronicity through targeting cytokine and NO generation by infected macrophages. Experimental Leishmania infection The role of T cells and their cytokines in determining disease outcome in leishmaniasis has been investigated extensively3,6,7. In mice, the inability to control leishmaniasis has been correlated with the absence of IFN-g production by parasite-specific T cells and their failure to activate macrophages to destroy intracellular amastigotes3,6. Macrophage-derived tumor necrosis factor a (TNF-a), the oxidative burst, and/or functional inducible NO synthase (iNOS or type II NOS) seem to be essential for parasite elimination by these cells8. The human immune response against Leishmania infection has been documented by various authors4,7. Experimental models clearly indicate a role for specific Leishmania antigens in inducing immune responses from both CD41 and CD81 T cells4,9–12. IFN-g, lipopolysaccharides and/or TNF-a4,5,7,13,14 can enhance the in vitro

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Parasitology Today, vol. 15, no. 8, 1999

Focus

Correlating in vivo and in vitro findings Analysis of cytokine expression during human cutaneous or mucocutaneous leishmaniasis4,8,17,18,22–24, although mostly limited to restricted endemic areas, showed a good relationship between in vivo cytokine expression, disease progression, and the effect of the same cytokines on leishmanicidal activity of human macrophages (Fig. 1). Our studies suggest that two independent pathways may promote leishmanicidal activity of infected human macrophages in vitro: (1) via the IFN-g receptor, and (2) CD23 ligation5. Both pathways are able to induce TNF-a and NO production by infected human macrophages, two factors involved in L. major killing by these cells. However, TNF-a alone failed to induce direct in vitro killing of L. major by macrophages5. When macrophages were stimulated with IFN-g or anti-CD23 alone, the addition of recombinant IL-4 or IL-10 significantly reduced their leishmanicidal activity in vitro21 (Fig. 1b). Interestingly, macrophage activation through both CD23 and IFN-g pathways protected them from the inhibitory effect of IL-4 and/or IL-10 (Ref. 21). These data suggest that inhibitory cytokines cannot eliminate effective leishmanicidal activity of human Parasitology Today, vol. 15, no. 8, 1999

Infection

IFN-g

TNF-a

CD23ÐIgE

Healing

IL-4 Infection

CD23 and the leishmanicidal activity of human macrophages IgE synthesis and CD23 expression are altered during parasitic infections, although the mechanisms involved are not known. Various data16–18 now show rapid in vivo expression of the gene encoding interleukin 4 (IL-4) during antimicrobial activity. IL-4, as well as IL-13, promotes IgE gene expression, while expression of CD23 mRNA by human cells is strongly induced by IFN-g, IL-4 and IL-13 and requires the activation of Janus kinases and Stat6 DNA-binding activity (reviewed in Ref. 19). CD40 ligation also induces CD23 gene expression, in part through the activation of a member of the TNF-a receptor-associated factor (TRAF) family19. In an in situ analysis of inflamed skin during cutaneous leishmaniasis17, we have detected relatively high CD23 gene expression compared with normal human skin. The b isoform of the CD23 antigen belongs to the c-type lectin family and is an activation antigen expressed by human, monkey and rat macrophages, but it has not been detected in mice. Like several important surface antigens in infectious disease, the CD23 lectin domain seems to react with fucose and mannose. The crosslinking of CD23 by IgE and anti-IgE immune complex (IgE-IC) or by specific monoclonal antibodies (mAbs) (reacting with the same epitope as IgE) has been shown to induce proinflammatory responses in human and rat macrophages19. CD23stimulated cells produce TNF-a, IL-6, IL-1, thromboxane B2 and various oxygen intermediates20. In Leishmaniainfected macrophages, addition of IgE-IC or anti-CD23 mAbs resulted in the elimination of most intracellular L. major, L. infantum or L. braziliensis5,21. A similar but less potent effect was observed with infected cells treated with IFN-g. In addition, CD23 stimulation of uninfected cells protected against subsequent contamination with L. major, while pretreatment with IFN-g had no such effect5.

a

Intensity of expression

leishmanicidal activity of human monocytes/macrophages. However, the exact mechanism of Leishmania killing has not been clearly identified and, until recently, it was unclear whether NO was involved in human macrophage functions15.

Healing IL-10

Acute immune response

Long-term immune response

Disease progression

b IFN-g IFN-g IL-10 Cytokines added to CD23ÐIgE CD23ÐIgE CD23ÐIgE IFN-g IL-4 IL-4 IL-4 infected TNF-a IL-10 TNF-a cells TNF-a NO High Moderate Low Moderate release Parasite killing

80Ð100%

65Ð85%

12 months), the levels of IFN-g decrease while the levels of IL-10 increase. Dashed lines represent the evolution of cytokine expression during healing. The regulation of in vitro Leishmania major killing (% parasite elimination) and NO generation (levels of nitrites) by various factors added to infected human macrophages, compared with untreated cells and based upon data from Refs 5 and 21 (b). Each cytokine combination mimicked an in vivo period as indicated by the dashed arrows. IFN, interferon; IL, interleukin; NO, nitric oxide; TNF, tumor necrosis factor.

macrophages when both synergistic IFN-g and CD23 pathways have been activated. This correlates exactly with in vivo cytokine patterns observed during effector immune responses before healing or during delayedtype hypersensitivity (Fig. 1a). In vitro, in the absence of IFN-g, the combination of IL-4, IL-10, IgE–antigen and TNF-a failed to induce Leishmania clearance in infected human macrophages21 (Fig. 1b), correlating with in vivo expression of these factors during disease exacerbation (Fig. 1a). These data suggest that active disease may be related to high IL-10 and low IFN-g levels in vivo, while high IL-4 levels did not always correlate with disease progression16–18. Therefore, paradoxical continuous in vivo expression of IL-4 during Leishmania infection may 343

Focus Parasites

Macrophages B cell IL-4 T cell IgE

IFN-g IL-4

FceRIIÐCD23

TNF-a NO TNF-a

+ IFN-g IL-12 TNF-a Anti-ROI

X Ð

X

X

X

Parasite killing

IL-10 IL-4 . O2 TGF-b

Fig. 2. Model of the human immune response network to Leishmania infection. This gives IL-4 and B cells a role during the anti-leishmanial response of macrophages. TNF-a and NO seem to play an effector role and may be down- or upregulated by various factors. IFN, interferon; IL, interleukin; NO, nitric oxide; ROI, reactive oxygen intermediates; TGF, transforming growth factor; TNF, tumor necrosis factor.

be explained by its opposing effects: inducing leishmanicidal activity indirectly through IgE and CD23 induction, or directly inhibiting parasite killing by decreasing NO generation. Is NO involved? Among oxidants, NO has emerged as an important cytotoxic and cytostatic mediator for various intracellular parasites, including most Leishmania strains1,3,25. NO is generated from the oxidation of the terminal guanidonitrogen atom(s) of L-arginine by NADPH-dependent enzymes, the NOSs26. NO involvement in the leishmanicidal activity of mouse macrophages leads to the question of whether NO functions in CD23-mediated parasite killing. Using CD23 crosslinking through its precise IgE-binding epitope, L-arginine-dependent NO generation was observed in human macrophages, eosinophils and epithelial cells, as well as in rat macrophages19,20. Of interest, another member of this protein family CD69 was also shown to mediate NO induction in human monocytes27. However, CD23-mediated NO generation by human macrophages is downregulated by various endogenous parameters, namely: (1) low surface CD23 density; (2) high levels of IL-10 or transforming growth factor b (TGF-b) production; and (3) cell activation before CD23 ligation. Nevertheless, in contrast to most earlier studies, our data suggest the involvement of the L-arginine–NO pathway in Leishmania killing by human macrophages for the following reasons: (1) addition of the NOS inhibitor, N G -monomethyl-L-arginine, reversed leishmanicidal 344

activity; (2) the protective effect of CD23 crosslinking correlated with NO generation; and (3) chemical NO donors induce extracellular and intracellular killing of cutaneous leishmaniasis species5,21. Furthermore, increased expression of physiological NO inhibiting factors, such as IL-10, TGF-b and IL-4, is observed during disease progression7,17,18,23,24 (Fig. 2). Apart from NO derivatives, superoxides seem to have a minor role (if any) during in vitro killing of L. major by macrophages. This is suggested by the inability of superoxide dismutase (SOD) to reverse Leishmania killing by infected human or murine macrophages5,28. Treatment with SOD even increased NO production in macrophages, perhaps as a result of inhibition of interactions between O2– and NO, which are known to reduce intracellular NO levels. The presence of superoxides might also result in the generation of peroxynitrites, potent toxic and proapoptotic agents that may kill macrophages26 rather than parasites. Chronic exposure to peroxynitrites might also play a role in ulceration and disease progression. Finally, preliminary analysis during delayed-type hypersensitivity to Leishmania antigens suggested that the NOS-II protein is produced in situ, but the nature of NOS-II-synthesizing cells remains to be clarified (I. Vouldoukis, unpublished). Therefore, parasite killing seems to involve a balanced network of multiple cells and mediators, as is shown in Fig. 2. Implications for treatment These in vivo and in vitro observations might clarify the role of IL-4, IgE and CD23 during cutaneous leishmaniasis, and to some extent during other protozoan infections. Data discussed here also indicate a role for NO and its regulation during the antimicrobial activity of human macrophages. Therapeutic approaches might exploit this NO pathway29, or directly ligate CD23 by appropriate counterstructures19. The latter approach would allow targeting of the macrophage response and prevent nonspecific NO-mediated toxicity. In vivo studies are now in progress using chemical NO donors and/or CD23 ligands to obtain an appropriate therapeutic approach for leishmaniasis. Acknowledgements We thank Geoffrey A.T. Target for kindly reviewing our work; and Otamires A. da Silva, Andre F. Furtado and Murizio L. Jardim for their contribution to the in vivo studies. References 1 Liew, F.Y. et al. (1990) Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J. Immunol. 144, 4793–4802 2 Green, S.J. et al. (1990) Activated macrophages destroy intracellular Leishmania major amastigotes by an L-argininedependent killing mechanism. J. Immunol. 144, 278–286 3 Stenger, S. et al. (1994) Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major. J. Exp. Med. 180, 783–793 4 Reed, S.G. and Scott, P. (1993) T-cell and cytokine responses in leishmaniasis. Curr. Biol. 5, 524–531 5 Vouldoukis, I. et al. (1995) The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the FceRII/CD23 surface antigen. Proc. Natl. Acad. Sci. U. S. A. 92, 7804–7808 6 Reiner, S.L. and Locksley, R.M. (1995) The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13, 151–177 7 Modlin, R.L. and Nutman, T.B. (1993) Type 2 cytokines and negative regulation in human infections. Curr. Opin. Immunol. 5, 511–517 8 Bogdan, C. et al. (1996) Invasion, control and persistence of Leishmania parasites. Curr. Opin. Immunol. 8, 517–525

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Focus 9 Skeiky, Y.A.W. et al. (1995) A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Thl-type cytokine profile and to produce interleukin12. J. Exp. Med. 181, 1527–1537 10 Mougneau, E. et al. (1995) Expression and cloning of a protective Leishmania antigen. Science 268, 563–566 11 Campos-Neto, A. et al. (1995) Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages. J. Exp. Med. 182, 1423–1433 12 Smith, L.E., Rodrigues, M. and Russell, D.G. (1991) The interaction between CD8+ cytotoxic T cells and Leishmania-infected macrophages. J. Exp. Med. 174, 499–505 13 Murray, H.W., Rubin, B.Y. and Rethermel, C.D. (1983) Killing of intracellular Leishmania donovani by lymphokine-stimulated human mononuclear phagocytes. Evidence that interferon g is the activating lymphokine. J. Clin. Invest. 72, 1506–1510 14 Barral-Netto, M. et al. (1991) Tumor necrosis factor/cachectin in human visceral leishmaniasis. J. Infect. Dis. 163, 853–857 15 Denis, M. (1994) Human monocytes/macrophages: NO or no NO? J. Leukocyte Biol. 55, 682–684 16 Scott, P. et al. (1996) Early IL-4 production does not predict susceptibility to Leishmania major. Exp. Parasitol. 84, 178–187 17 Vouldoukis, I. et al. (1994) CD23 and IgE expression during human immune response to cutaneous leishmaniasis: possible role in monocyte activation. Res. Immunol. 145, 17–27 18 Coutinho, S.G. et al. (1996) T-cell responsiveness of American cutaneous leishmaniasis patients to purified Leishmania pifanoi antigens and Leishmania braziliensis promastigote antigens: immunologic patterns associated with cure. Exp. Parasitol. 84, 144–155

19 Mossalayi, M.D., Arock, M. and Debré, P. (1997) CD23/FceRII: signaling and clinical implication. Int. Rev. Immunol. 16, 129–146 20 Dugas, B. et al. (1995) Nitric oxide production by human monocytes: evidence for a role of CD23. Immunol. Today 16, 574–580 21 Vouldoukis, I. et al. (1997) Interleukin-10 and interleukin-4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation. Eur. J. Immunol. 27, 860–865 22 Primez, C. et al. (1993) Cytokine patterns in the pathogenesis of human leishmaniasis. J. Clin. Invest. 91, 1390–1395 23 Melby, P.C. et al. (1996) In situ expression of interleukin-10 and interleukin-12 in active human cutaneous leishmaniasis. FEMS Immunol. Med. Microbiol. 15, 101–107 24 Barral, A. et al. (1993) Transforming growth factor-b as a virulence mechanism for Leishmania braziliensis. Proc. Natl. Acad. Sci. U. S. A. 90, 3442–3446 25 Nussler, A. and Billiar, T.R. (1993) Inflammation, immunoregulation, and inducible nitric oxide synthase. J. Leukocyte Biol. 54, 171–178 26 Moncada, S. and Higgs, E.A. (1993) The L-arginine–nitric oxide pathway. New Engl. J. Med. 329, 2002–2012 27 De Maria, R. et al. (1994) Triggering of human monocyte activation through CD69, a member of the natural killer gene complex family of signal transducing receptors. J. Exp. Med. 180, 1999–2004 28 Assreuy, J. et al. (1994) Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major. Eur. J. Immunol. 24, 672–676 29 Lopez-Jaramillo, P. et al. (1998) Treatment of cutaneous leishmaniasis with nitric-oxide donor. Lancet 351, 1176–1177

Letters Nested PCR for the Detection of Cryptosporidium parvum In their recent review, Morgan and Thompson1 described the pitfalls of using nested PCR for the detection of the diarrhoea-causing protozoan, Cryptosporidium parvum, in faecal samples or in waste or surface water. In addition to inhibition of the polymerase enzyme by bile constituents in clinical specimens, and by algal and inorganic material in environmental samples, nested PCR is also very susceptible to false positives due to contamination and carry-over. We agree with the reservations of Morgan and Thompson regarding the need for strict attention to eliminate contamination, and note that nested PCR may be extremely useful in some circumstances, if such precautions are made. Recently, we have successfully used PCR, specifically targeted to the dihydrofolate reductase (DHFR) gene2, where microscopy failed to detect low levels of C. parvum oocysts in an HIV-positive patient undergoing anti-retroviral therapy3. A nested PCR protocol here improved the assay sensitivity. Nested PCR may also have application under the controlled conditions of the research laboratory. For example, the DHFR-based typing system mentioned above should prove to be a useful marker Parasitology Today, vol. 15, no. 8, 1999

for genotypic analysis of C. parvum isolates from different hosts. Many workers devising PCR assays for detecting and genotyping this parasite have chosen the multicopy rRNA genes to increase sensitivity1. However, the heterogeneity of the rDNA transcription units in C. parvum reported recently4 may limit their suitability for isolate genotyping. A single-copy housekeeping gene, such as DHFR5, would not be affected by such heterogeneity, as only one genotype-specific profile is possible for each parasite. So far, we have employed the DHFRbased protocol to genotype over 20 C. parvum isolates (from Europe, North America, Africa and Asia) from human and from bovine hosts. As both C. parvum and C. muris may be isolated from infected calves6, we believed it was important to develop an assay capable of detecting C. parvum but not C. muris, especially when water sources are contaminated with low numbers of unidentified cryptosporidial oocysts. The primer sets we have designed do not amplify the DHFR gene from a number of calf C. muris isolates and, as such, may provide useful information for epidemiologists investigating the outbreak potential of cryptosporidial oocysts detected in water supplies.

With careful handling, nested PCR is a useful tool for the detection and typing of C. parvum and should be investigated further. Because of its extreme sensitivity, it may be particularly useful in circumstances where reliable and sensitive detection of low numbers of C. parvum oocysts is needed (eg. for monitoring oocysts in backwash water7). Furthermore, coupling this protocol with new technologies may result in the development of enhanced detection systems capable of providing simultaneous genotypic, viability and quantitative information. References 1 Morgan, U.M. and Thompson, R.C.A. (1998) Parasitol. Today 14, 241–245 2 Gibbons, C.L. et al. (1998) Parasitol. Int. 47, 139–147 3 Miao, Y.M. et al. AIDS (in press) 4 Le Blanq, S.M. et al. (1998) Mol. Biochem. Parasitol. 90, 463–478 5 Vasquez, J.R. et al. (1996) Mol. Biochem. Parasitol. 79, 153–165 6 Anderson, B.C. (1998) J. Dairy Sci. 81, 3036–3041 7 Gibbons, C.L. et al. (1998) Protist 149, 127–134 Cindy L. Gibbons Fatih M. Awad-El-Kariem Department of Biology Imperial College of Science, Technology and Medicine BMS Building Exhibition Road London UK SW7 2AZ

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