The effect of the lack of histamine and histamine H4R on the functional properties of dendritic cells.

Transcription

1 The effect of the lack of histamine and histamine H4R on the functional properties of dendritic cells. PhD Thesis Ivett Jelinek Semmelweis University Molecular Medicine Doctoral School Supervisor: Dr. Valéria László associate professor, Ph.D. Opponents: Dr. Tíema Berki associate professor, Ph.D. Dr. Beáta Dérfalvi research assistant, Ph.D. President of the Doctoral Rigolosum: Prof. Dr. Imre Oláh, Ph.D. Members of the Doctoral Rigolosum: Dr. Árpád Lányi senior lecturer, Ph.D. Dr. András Kiss senior lecturer, Ph.D. Budapest 2007

2 Introduction Dendritic cells (DCs) are the most potent antigen presenting cells specialized for capture, uptake, transport, processing and presentation of antigens (Ag) to T-cells, so they play an important role in both innate and adaptive immunity against pathogens. In peripheral tissues, "immature" DCs take up self and nonself antigens. After activation by danger signals together with inflammatory agents such as PAMPs (Pathogen-Associated Molecular Pattern), endogenous pro-inflammatory compounds like inflammatory cytokines (IL-1 or TNF- α) or bacterial products such as LPS, DC "mature" by increasing their expression of MHC class II and co-stimulatory molecules (CD40, CD80 and CD86). DC maturation and consequently DC function are strongly influenced by other inflammatory mediators such as histamine, which is in the focus of this thesis. Maturing DC then migrate to the T cell zones of secondary lymphoid organs where they present Ag and co-stimulatory signals to recirculating T lymphocytes and induce appropriate immune responses. Histamine, well known as a mediator of acute inflammatory and immediate hypersensitivity responses, is a molecule with a broad spectrum of functions. It exerts various immunomodulatory effects and is involved in many regulatory processes, especially in the regulation of antigen-specific immune responses. Therefore, histamine plays affects a variety of cellular functions, particularly in cells of the immune system. Since all cells that are important for the regulation of immune responses express histamine receptors on their surface and are able to produce and secrete histamine, this small molecule participates in the regulation of the immune system in various ways, but especially by modulating the cytokine profile and the functions of antigen presenting cells. Thus, histamine directly affects dendritic cell function, but also the functionality of monocytes, B-cells and T-cells, in which it especially influences the Th1/Th2-regulation. Histamine was proved to influence migration in several types of leukocytes, using different histamine receptors for 1

3 mediating this effect. Histamine H4 receptor (H4R) seems to play a special role, as experiments provided direct proof that triggering of H4R induces chemotaxis of eosinophils and mast cells. In this thesis, the involvement of histamine in the regulation of some crucial DC functions was examined. DCs were isolated from spleens of two different knock-out mouse models, a histamine-free mouse model lacking histidine decarboxylase (HDC), the enzyme solely responsible for histamine synthesis, and another mouse model lacking one of the four receptors for histamine, the recently discovered H4R, which was suggested to play a pivotal role in immune regulation. By the use of these isolated DCs, the effects of histamine on DCs, especially mediated through H4R, were investigated, focusing on three processes important for DC function: antigen presentation, T- cell polarization and migration. Since most data about the influence of histamine on DCs were generated by performing experiments using in vitro differentiated DCs, results from spleen-derived DCs are appropriate models for investigating the effects of histamine on the functional properties of dendritic cells and represent promising tools for the analysis of this question.. 2

4 Aims 1. Investigation of the role of histamine in DC antigen presentation, the most crucial function of DCs. 2. Analysis of DC cell surface markers for elucidating possible effects of histamine on the distribution of DC subpopulations and on the expression of co-stimulatory molecules. 3. Investigation of the role of histamine in DC cytokine production in vitro. Evaluation of the hypothesized effects of histamine on the regulation of DC-mediated T-cell polarization. 4. Investigation of the role of histamine in DC cytokine production in vivo. Evaluation of the hypothesized effects of histamine on the regulation of DC-mediated T-cell polarization 5. Examination of the expression of different histamine receptors on mouse DCs. 6. Investigation of the importance of H4R in histamine-mediated alteration of DC antigen presentation capacity. 7. Elucidation of the function of H4R in DC cytokine production in vitro. 8. Investigation of the effects of H4R triggering on DC migration, a function necessary for controlled movement between periphery and lymphoid organs. 3

5 Methods Mice: For our experiments we used two knock out mouse models, namely histidine-decarboxylase knock out (HDC -/- ) and H4R knock out (H4R -/- ) animals. In all experiments, wild type mice on the same background were used for comparison purposes. Cells: Splenic DCs: DCs were isolated from HDC -/-,H4R -/- or wild type mouse spleen using the MACS technique. CTLL-2: Lymphoblast cell line; this line is a cytotoxic T-cell clone derived from a C57BL/6 mouse. Cells are dependent upon IL-2 for growth, and can be used for assaying IL-2. 5/4E8: T cell hybridoma cell line, specific for the human aggrecan peptide. In vitro antigen presentation assay: HDC -/- and H4R -/- and wild type DCs were cocultured for 24 hours with the T cell hybridoma cell line 5/4E8 specific for the human aggrecan peptide. Then the supernatants were assayed in a CTLL-2 proliferation assay using MTT. In these experiments we used human aggrecan peptide as antigen. This peptide was presented by DCs to the specific T cells, and the efficacy of the antigen specific T-cell stimulation was determined by measuring the amount of produced IL-2. In some experiments, wild type DCs were treated with specific H4R antagonists (JNJ , 10-6 M) before the experiments. In vitro DC stimulation: Wild type, HDC -/- and H4R -/- DCs were treated with 1μg/ml LPS and/or 10-6 M L-histamine for 24 hours. In a second experiment, LPS-activated wild type DCs were further treated with 10-6 M L-histamine and/or 10-6 M JNJ Then the cells were collected and used for measuring the expression of different DC cytokines, such as IL-2, IL-10, IFNγ and IL-12 by real time PCR. Finally, DC culture supernatants were used for evaluation of the protein levels of IFNγ and IL-12. In vivo DC stimulation: Wild type and HDC -/- mice were injected intraperitoneally with either 0.2 ml PBS or 0.2 ml Complete Freund's Adjuvant 4

6 (CFA) emulsion. After 9 days of immunization mice were killed and spleens were removed for DC isolation. Isolated spleen DCs were used for RNA preparation followed by real time PCR analysis. In vitro migration assay (Transwell system): Transwell filters were prepared by incubation of the transwells with RPMI medium. Then, chemoattractants were added to the lower chamber (100ng/µl CCL19 and/or 10-6 M L-histamine). Medium from the upper chamber was removed and replaced by wild type and H4R -/- DCs. Migration was performed for 90 minutes. The number of migrated DCs was calculated using a normalization method with microbeads followed by flow cytometric analysis. Similar experiments were performed using wild type DC pretreated with 10-6 M JNJ In vivo migration assay (FITC painting): The abdominal parts of wild type and H4R -/- mice were painted with FITC-dibutylphthalate-aceton solution, and 18 hour later the proportion of migrated FITC positive LC were measured in the draining lymph nodes by flow cytometry. 5

7 Results Dendritic cells have been well documented to be the most potent antigen presenting cells, therefore we compared the antigen-specific T cell stimulation capacity of wild type and HDC -/- DC by an in vitro antigen presentation assay. Using this system we were able to establish a very sensitive assay because as few as 10 3 DC/well were able to effectively stimulate /4 T cells to produce easily detectable amounts of IL-2 (p<0,001). In this assay we found a remarkable difference in the antigen presenting capacity of wild type and HDC - /- DC, as HDC -/- DC stimulated T cells more effectively than their wild type counterparts did (p=0,002). It is well known that distinct DC subpopulations exhibit different abilities in regulating the Th cell-mediated immune responses. However, there was no difference between wild type and HDC -/- mice either in the total number of spleen DC (CD11c+, MHCII+ cells) or in their surface marker expression patterns typical for the main DC subpopulations. Similarly, no difference was found in the surface expression of the predominant DC costimulatory molecules (CD80, CD86, CD40). As DC-produced cytokines play a critical role in T cell polarization, we analyzed the question whether an altered cytokine production could stand behind the altered antigen presenting capacity of the HDC -/- dendritic cells. In our in vitro studies we observed elevated IFNγ expression at both the mrna (p=0,015) and protein levels (p=0,007) in histamine free DCs and therefore these cells mediated stronger Th1 polarization. Knowing the limitations of in vitro experimental systems we performed an in vivo assay to measure the cytokine production of DCs. Our in vivo CFA stimulation experiment was able to confirm our in vitro results, as in this case the stimulated histamine free DCs produced higher levels of both IL-12 (p=0,015) and IFNγ (p=0,023). Interestingly, the levels of the immunosuppressive cytokine IL-10 were found to be also up-regulated in unstimulated HDC -/- DCs (p<0,001). However, after stimulation, in contrast to 6

8 Th1 cytokines, the level of IL-10 was down-regulated in histamine free DCs (p=0,008), but no such effect was seen in the wild type cells. In the second part of our investigations we made an attempt to identify the histamine receptor(s) mediating these effects. DCs are known to express at least three different histamine receptors, H1R, H2R and the novel H4R. In line with literature data, we also were able to detect the expression of these three receptor types in our system. In our next experiments, we studied the involvement of H4R in the mediation of previously discussed histamine effects. In these experiments we studied DCs, which either do not have H4R, namely H4R -/- DCs, or we blocked the H4R functions with the specific H4R antagonist JNJ After performing the antigen presentation assay we found again increased antigen presentation in H4R -/- DCs (p<0,001), as previously in the histamine free situation. We also obtained the same results when we blocked the H4R with its antagonist (p=0,003), but when we inhibited the other two histamine receptors we did not see any change in the efficacy of DC-mediated antigen presentation. Next we examined the role of H4R in DC cytokine production. Using LPS stimulated DCs, in the case of IFNγ we observed a trend that the H4R antagonist was able to neutralize the histamine-mediated decreasing effect on IFNγ expression, while in the case of IL-10, the blockade of H4R had no effect. In the case of IL-12 we were not able to observe any effect. DCs are typical migratory cells. A controlled migration is essential for fulfilling their functions, since antigen uptake and antigen presentation take place on different sites in the body. We found decreased expression of CCR7 in H4R -/- DCs compared to wild type cells (p=0,04). Therefore we performed an in vitro transwell migration assay, and we found decreased migratory capacity of H4R -/- DCs (p=0,028). These results were confirmed by using the H4R antagonist, as well. 7

9 Finally we checked the migratory potential of DCs in an in vivo experiment, too, using the so-called FITC painting assay. In this assay we found again decreased migration of DCs in H4R -/- mice (p=0,013), so we concluded that H4R is essential for DC migration. 8

10 Conclusion Histamine is well-known about its different roles in the immune system. In this thesis we examined the possible effects of histamine on DCs, because DCs are the main antigen presenting cells and it is highly probable that histamine (produced both by other cells and by DCs themselves) gets in close connection with DCs in the tissue environment. Focusing on antigen presentation, T-cell polarization and migration, the role of histamine in the regulation DC functions was examined. By the use of DCs isolated from mouse spleen of histamine-free (HDC -/- ) and histamine H4 receptor deficient (H4R -/- ) mouse models, the effects of histamine on these functions in general, but especially when mediated through H4R, was investigated. Performing antigen presentation assays with DCs from both knock-out mouse models, and using the specific H4 receptor antagonist JNJ , we demonstrated that histamine has an inhibiting effect on DC antigen presentation capacity, which is primarily mediated through H4R. Furthermore, surface marker analysis revealed that the increased antigen presentation by HDC -/- DCs is neither due to changes in the distribution between the major functional subpopulations, nor a consequence of an altered co-stimulatory molecule profile on these cells. Investigation of the effect of histamine on T-cell polarization by DCs revealed that histamine has an important role in the regulation of the cytokine production by DCs. Our data showed that DCs activated to antigen presentation display a rather Th1-polarized cytokine profile in HDC -/- mice. Further analysis revealed that the inhibitory effect of histamine on mrna expression of cytokines is only partly mediated by H4R, as histamine might also exert some of its regulatory effects via H1R and / or H2R, or via a combination of H1R, H2R and H4R. The influence of H4R-mediated signals on DC migration was also found to be highly important, as the lack of H4R resulted in significant decrease in the migration capacity of DCs both in vitro and in vivo. 9

11 Taken together, histamine was found to modulate antigen presentation, T cell polarization, cytokine production, and migration of DCs, and these effects are mediated mainly, but not completely by H4R. As a conclusion, we found that histamine plays an essential role in DC functions, and H4R is crucial in mediating of these effects. These observations suggest new potential applications for the novel H4R ligands in vaccination therapy of several immun-related disorders, such as allergy or cancer. 10

12 Publications Publications related to this work: 1. Jelinek I, Laszlo V, Buzas E, Pallinger E, Hangya B, Horvath Z, Falus A. Increased antigen presentation and T(h)1 polarization in genetically histamine-free mice. Int Immunol. 2007;19: IF: 3, Buzas EI, Gyorgy B, Pasztoi M, Jelinek I, Falus A, Gabius HJ. Carbohydrate recognition systems in autoimmunity. Autoimmunity. 2006;39: IF: 1,49 3. Kis Z, Pallinger E, Endresz V, Burian K, Jelinek I, Gonczol E, Valyi- Nagy I. The interactions between human dendritic cells and microbes; possible clinical applications of dendritic cells. Inflamm Res. 2004;53: IF: 1,45 Other publications: 1. Horvath Z, Pallinger E, Horvath G, Jelinek I, Falus A, Buzas EI. Histamine H1 and H2 receptors but not H4 receptors are upregulated during bone marrow regeneration. Cell Immunol Dec;244(2): IF: 1,558 11

13 Acknowledgements Hereby I would like to thank to everyone who took part in the preparation of this PhD thesis, regardless what kind of help his or her contribution actually meant. First of all to everyone in my family, because no question it would be completely impossible for a PhD student to finish such a work without the patience, endurance, and everyday support of his family. Second, I am very thankful to my supervisors, who led my steps during the last four years. They helped me many ways; with their knowledge, experience, know-how, with their well-written grants, and last but not least by spending their quite limited time with my ideas. I am very thankful to András Falus, Valéria László, Edit Buzás, Martin Zenke and Thomas Hieronymus for their help. Third, my acknowledgements are due to all my friends, with whom I had the opportunity to work together during these years. I have met many PhD students, technicians and postdocs in this time, as I shared a computer, worked in the same lab or on the same project with someone. I would like to thank for the many excellent ideas, fine proposals, helping hands, and evil questions to Zoltán Wiener, Krisztina Hegyi, Márton Keszei, Zsuzsanna Horváth, Dániel Erdélyi, Viktor Molnár, Mária Pásztói, Anna Gilicze, Orsolya Láng, Eszter Lajkó, Péter Pócza, Ildikó Ungvári, Ágnes Semsei, Gergő Tölgyesi, Melinda Rácz, Piritta Jäntti and Steffen Gräber. Finally, I would like to express my sincere thanks for all my colleagues I worked together at the Semmelweis University, Department of Genetics Celland Immunobiology, and in the Institute for Biomedical Engineering, Department of Cell Biology at the University Hospital of Aachen. I am very thankful to them for accepting me as part of their community, and for their open-hearted collegiality. Many people in these institutes played important roles in this work: I have to thank the most to Valéria László for her excellent supervision and for her kindness and comprehension, to Éva Pállinger for her excellent introduction in the techniques of flow cytometry, and to Krisztina Farkasné Nagy for her introduction to cell cultivation. 12