Human dendritic cells : a study of early events during pathogen recognition and antigen endocytosis

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M A L M Ö U N IV E R S IT Y H E A LT H A N D S O C IE T Y D O C T O R A L D IS S E R TA T IO N 2 0 0 9 :4 P E T E R H E L L M A N M A L M Ö U N IV E R

PETER HELLMAN

HUMAN DENDRITIC CELLS

A study of early events during pathogen recognition

and antigen endocytosis

isbn/issn 978-91-7104-231-6/ 1653-5383 H U M A N D E N D R IT IC C E L L S

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H U M A N D E N D R I T I C C E L L S A S T U D Y O F E A R L Y E V E N T S D U R I N G P A T H O G E N

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Malmö University

Health and Society Doctoral Dissertations 2009:4

Cover: ”Dendritic” Derek Beggs Ba Hons © http://www.derekbeggs.com. Reproduced with permission from the artist.

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PETER HELLMAN

HUMAN DENDRITIC CELLS

A study of early events during pathogen recognition

and antigen endocytosis

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ABBREVIATIONS

APC Antigen presenting cell BDCA Blood dendritic cell antigen CCP Clathrin coated pit

CCV Clathrin coated vesicle CD Cluster of differentiation

CDP Common dendritic cell progenitor CLA Cutaneous lymphocyte antigen CLR C-lectin receptor

CMP Common myeloid progenitor CpG Cytosine phosphate guanosine DC Dendritic cell

ER Endoplasmic reticulum FcR Fc receptor

Flt3 FMS-related tyrosine kinase 3 receptor Flt3L Flt3 ligand

CSF-1 Colony stimulating factor-1 CSF-1R CSF-1 Receptor

GM-CSF Granulocyte and macrophage colony stimulating factor GMP Granulocyte and macrophage progenitor

HEV High endothelial venule HCV Hepatitis C virus

HPC Hematopoietic progenitor cell HSC Hematopoietic stem cell IC Immune complex IFN Interferon Ig Immunoglobulin IL Interleukin

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LC Langerhans cell

LN Lymph node

LPS Lipopolysaccharide LTA Lipoteichoic acid

ODN Oligodeoxyribonucleotide

MDP Macrophage and dendritic cell progenitor MHC Major histocompatibility complex

MLR Mixed-lymphocyte reaction MoDC Monocyte derived dendritic cell MPS Mononuclear phagocyte system

NADPH Nicotinamide adenine dinucleotide phosphate, reduced form PAMP Pathogen associated molecular patterns

PBMC Peripheral blood mononuclear cell pDC plasmacytoid dendritic cell

PLGA Poly (lactic-co-glycolic acid) PRR Pathogen recognition receptor SLO Secondary lymphoid organ SR Scavenger receptor

TLR Toll-like receptor TNF Tumour necrosis factor VLP Virus like particle

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ABSTRACT

The mononuclear phagocyte cell system includes monocytes, macrophages and dendritic cells which are important cells in order to recognize, ingest, destroy and also present part of a pathogen to T-lymphocytes in order to activate the adaptive immune system. Dendritic cells (DCs) stand out in their ability to stimulate T-lymphocytes and are also believed to be important to keep tolerance for “self-antigens”.

Therefore DCs are of interest for use in immunotherapy studies. However in most such studies to date, DC-like cells have been used, so called monocyte derived dendritic cells (moDCs).

The aim of this thesis was to investigate the early events following in vitro

activation of highly purified human DCs. In the first study we observed that the production of IL-8 and down regulation of CD128b preceded surface expression of MHC class II and CD40, 80 and 86. We have in the following studies used and demonstrated the practical use of zeolite particles as ligand carriers with the purpose to study the uptake mechanisms deployed by phagocytes. We show the advantage of using zeolite particles, due to their ability to bind various types of ligands i.e. proteins, oligonucleotides, lipophilic, and hydrophobic molecules. In addition, we have adsorbed bio molecules in sequential steps, which demonstrates the potential of co adsorbing ligands e.g. for targeting a specific endosomal compartment together with molecules sensing the endosomal microenvironment.

Coating zeolite particles with different biomolecules might provide further understanding of mechanisms involved in antigen sorting into endocytic compartments.

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LIST OF PAPERS

I. Hellman P. and Eriksson H. Early activation markers of human peripheral dendritic cells. Human Immunology 68, 2007

II. Andersson L., Hellman P. and Eriksson H. Receptor-mediated endocytosis of particles by peripheral dendritic cells. Human Immunology 69, 2008 III. Hellman P., Andersson L. and Eriksson H. Ligand surface density is

important for efficient capture of immunoglobulin and phosphatidylcholine coated particles by human peripheral dendritic cells. Cellular Immunology

258, 2009

IV. Hellman P., Andersson L., Dahm Å. and Eriksson H. Dealuminated zeolites as sensors of endosomal microenvironments and vehicles of antigen delivery.

Manuscript in preparation

The publications are reproduced with permission from the publishers.

Contribution to the publications

I performed most of the planning and essentially all experimental work in papers I, III and IV in particular concerning coating of particles with IgG, intracellular staining, flow cytometry analysis as well as writing the major part of the manuscripts. My contribution in paper II was the part regarding experimental work with cytokine analysis and LTA-pyrogen assay. I also took part in discussions of the results and the manuscript.

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INTRODUCTION

Bodily surfaces that are exposed to the surroundings carry inherent attributes that not only prevent intrusion but also gather information regarding the need of an adaptive immune response. The innate system consists of a humoral arm including antimicrobial peptides and complement proteins, as well as a cellular arm, which is mainly composed of cells known as phagocytes. Phagocytic mononuclear cells are sometimes referred to as the mononuclear phagocyte system (MPS) in which monocytes, macrophages and dendritic cells (DCs) are included.

DCs are a population of antigen presenting cells (APC) with a unique ability to induce proliferation of naïve T-lymphocytes thereby being able to link together the innate and adaptive immune system. DCs may also be important to induce immune tolerance against self and harmless environmental antigens and yet, in a highly specific way regulate an adaptive immune response. Immune responses, being of great potency, make this feature of selectivity and control highly critical.

Because of these unique features, DCs have been widely used in cancer biology and in transplantation and autoimmunity studies

When it comes to cancer biology, despite great efforts, many DC based immunotherapy trials to date, have not yielded data from which firm conclusions can be drawn (1). Thus, varying immunological and clinical results using DCs generated from CD34+ cells have been reported in melanoma patient studies (2-4). There are many reasons for this, including non-standard DC preparations, different maturation and vaccination regimes, and the use of different antigen preparations. Extensive animal studies have been conducted but optimal parameters in humans remain to be established.

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Understanding the immunobiology behind a certain pathological state, e.g.

cancer, in humans is much more difficult than in genetically well-defined objects such as inbred mice. In humans, cancer is a complex, long lasting metastatic disease that occurs in genetically dissimilar subjects. So, a main “translation” challenge for the future is the transfer of what we have learned from immunotherapy studies in the mouse model to humans. To be successful in such an attempt, a broadening of basic research in man is required (5).

As human dendritic cells are a heterogeneous and a sparsely distributed population, most experimental and clinical studies today rely on the in vitro

generation of DC-like cells, either from CD34+ progenitors or those derived from peripheral blood monocytes. The latter are widely used in immunotherapy studies because of the relative ease to obtain large quantities of cells, and there are numerous cytokine cocktails described in the literature used for the generation of monocyte-derived DCs (moDCs). The combination of GM-CSF with IL-4 was the first to be reported and is by far the most extensively utilized and characterized cytokine combination used for DC differentiation in vitro (6). GM-CSF appears to be required to support monocyte survival and differentiation in vitro; IL-4 on the other hand has been shown to induce DC differentiation of human monocytes by an inhibitory function on macrophage differentiation (7). Despite the extensive use of the GM-CSF / IL-4 combination, it is still debated whether or not this treatment reflects a natural pathway for DC generation (8).

Although laboratories worldwide are exploring clinical therapeutic applications for DCs, many aspects of DC-biology are still poorly understood. Therefore there is a significant need to study biological aspects of peripheral blood DCs and moDCs regarding such functions as antigen uptake, processing, and also how they can be induced to undergo maturation in response to pathogens or cytokine stimulation.

Taking into account ethical considerations and difficulties generating DCs from human lymphoid tissues, DCs from peripheral blood are the easiest accessible source when studying human DCs. Human peripheral blood DCs are a rare cell population that constitute about 1% of the peripheral blood mononuclear cells (PBMC) from healthy human individuals (9-11).

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Isolation of such DCs by flow cytometry sorting, or using magnetic beads, is efficient, however expensive, and relatively small numbers of cells can be obtained. As described above larger numbers of DC-like cells can be derived

by in vitro differentiation of peripheral blood monocytes from whole blood or

buffy-coat preparations. However, this approach is laborious and the subsequent differentiation towards DCs is time consuming, requiring 6–9 days. Another complicating factor in generating appropriate in vitro DCs is the numerous variants of DC populations in vivo (12).

In paper 1: initial phenotypic changes and cytokine production during early events in an in vitro activation model of human peripheral blood DCs was studied with the intent to explore the use of appropriate TLR ligands for the activation of each particular DC subpopulation. Efficient antigen uptake and subsequent T-lymphocyte stimulation could very well be dependent on initial events during DC activation.

In papers 2, 3 and 4: we have studied and elucidated ligand enhanced capture and endocytosis/phagocytosis of particles by human peripheral DCs. Due to the capacity to bind both hydrophilic and hydrophobic molecules without covalent attachment, and the possibility of sequential attachment of bio-molecules, zeolite particles were shown to be an excellent tool to study ligand enhanced endocytosis of particles.

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DENDRITIC CELLS

Definition and origin

DCs were originally described as a cell population enriched from mouse spleen, responsible for the so called “mixed-lymphocyte reaction” (MLR) (13). Today such splenic cells are known as conventional DCs, and are present in lymphoid organs. Conventional DCs have the DC form and function and can be divided into subsets according to phenotype, function and localization (14). Conventional DCs are either classified as migratory, these are the classical textbook DCs, acting as sentinels in peripheral tissues and will migrate to lymph nodes (LN) bearing antigens from the periphery, e.g. Langerhans cells (LC). Conventional DCs also include so called lymphoid-tissue-resident DCs,

e.g. thymic conventional DCs and splenic conventional DCs. Lymphoid-tissue resident DC do not migrate through the lymph and their function is to collect and present antigens in the lymphoid organs. For clarity, conventional DCs will hereafter be named DCs. Another category of DCs considered today is the so-called pre-DCs, the last precursor stage en route to DCs. In many cases, further development of pre-DCs requires a microbial or cytokine stimulus; a pre-DC example is the plasmacytoid cell (pDC) (15).

In the text below it should be considered that given the complexity of the DC network, most of the present knowledge today has been generated using whole-animal models.

Although the origin and renewal of macrophages and dendritic cells have been intensively investigated, lineage relation between these cell types are still unclear (16).

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Mononuclear cells develop from hematopoietic stem cells in the bone marrow via several commitment steps.

It is believed today that monocytes, several macrophage subsets, most of the DCs in the secondary lymphoid organs (SLO) of mice, and a minority of the DC population in the mouse thymus probably originate from a myeloid progenitor (16, 17).

An important hematopoietic growth factor receptor, expressed on primitive multipotent hematopoietic cells and monocytes, macrophages, and DCs is CSF-R (18-20). In mice lacking expression of CSF-1L or its receptor, several macrophage populations were absent, including osteoclasts, which resulted in the development of osteopetrosis in these mice (21). In earlier studies DCs were seen to reach normal numbers and therefore reported to be independent of CSF-1L (22). However, recent data indicate otherwise and CSF-1R expression was needed for the development of LCs (23) and when using a transgenic mouse model it was revealed that CSF-1R mRNA was expressed by DC subsets in several lymphoid organs and blood (24). Other cytokines like Flt3L, GM-CSF, and lymphotoxin-β are needed for development and homeostasis of macrophages and DCs (25-27).

In the prevalent model of DC and monocyte/macrophage development (Figure 1), intermediate progenitors first pass through a common myeloid progenitor (CMP), a granulocyte/macrophage progenitor (GMP), and the macrophage/DC progenitor (MDP) (17). CMP is reported to be heterogeneous in Flt3 expression, i.e. the receptor for the Flt3 ligand. It is the Flt3 expressing fraction of CMP that is able to produce DCs and pDCs found in mouse spleen and thymus (28, 29). The MDP has been shown to give rise to monocytes, several macrophage subsets, and spleen cDCs but no pDC precursor cells were detected (25, 30). However, recent data indicate that MDPs actually can give rise to pDCs in vivo (31).

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Figure 1. Current knowledge on the lineage relationships between macrophage and DC progenitors and the origin of macrophages and DC subsets. HSC: hematopoietic stem cell, CMP: common myeloid progenitor, GMP: granulocyte and macrophage progenitor, MDP: macrophage and dendritic cell progenitor, CDP: common dendritic cell progenitor, pDC: plasmacytoid cell. Adapted from (17).

Many cell fate decisions are involved in each of these differentiation steps that successively restrict the development potentials of the cells. A member of the ETS-family of DNA binding proteins, PU.1, is a transcription factor that has a regulatory role in many of these steps. PU.1 is expressed in hematopoietic cells and its deletion causes embryonic or neonatal death. In early myelopoiesis, PU.1 is reported to be important for inducing myeloid commitment steps in early multipotent progenitor cells (32, 33). There are several cell development options along the myeloid/monocytic-pathway that PU.1 controls by antagonistic interactions with transcription factors such as GATA-1, a hematopoietic transcription factor essential for normal megakaryopoiesis and erythropoiesis, and with GATA-2, needed for mast cell development (34).

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It is believed that when reaching the bipotent GMP stage, PU.1 is important for monocytic development (35).

Taken together, PU.1, controls several cell fate decisions during myelopoiesis and can block important regulators of other cell pathways. However, according to the literature, the antagonistic feature of PU.1 is not absolute but dependent on relative expression levels and the balance of specific transcription factors (34, 35).

This model is certainly not complete and additional transcriptions factors may be involved. The controversy regarding the origin of monocytes and DCs is far from resolved, and recently another precursor, the common DC precursor (CDP) was reported to generate DCs and pDCs, but no monocytes (36, 37).

Human blood dendritic cells

Human blood DC-subsets found in human peripheral blood are all characterized as HLA-DRhi cells that do not express markers of T-lymphocytes, B-cells, monocytes, or NK-cells, i.e. they are lineage negative, (lin-) cells (e.g., CD3, CD14, CD19, CD56 negative) (38, 39).

CD11c, an integrin often used as a myeloid marker that enables DCs to adhere to stimulated endothelium, is strongly expressed on three non-overlapping subsets found in human peripheral blood (39). O´Doherty et al., 1994 reported these classical CD11chi

DCs (40), and in paper I-IV we used the CD11c marker on isolated dendritic cells to indentify CD11chi

and CD11c

-cells in human peripheral blood. Hereafter, CD11chi

cells will be named myeloid DCs (mDCs). The CD11c- population constitutes the so-called pre-DCs, i.e. pDCs, vide supra. Mouse peripheral blood contains DC-subsets that are reported to be equivalents to human mDCs and pDCs (41).

Human mDCs have been further subdivided, based on the expression of CD16, CD1c, and blood dendritic cell antigen -3 (BDCA-3) (39). Amongst PBMCs, the major subsets of mDCs in human blood are the CD16+ and CD1c+ cells; the BDCA-3+ cells constitute only a minor population. On the basis of cytokine production the major mDC populations showed different responses after microbial stimulation.

Hence, CD1c+ cells produce high amounts of the chemokine IL-8 and are proposed to be involved in chemotaxis; the CD16+ cells produce preferentially TNF-α and are suggested to have a proinflammatory role (42).

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When the second main population of human peripheral blood DCs, pDCs, was first described, pDCs were thought to be the T-lymphocyte counterpart of plasma cells (43). Their plasmacytoid morphology, distinguished by a highly developed endoplasmic reticulum, was a clear indicator that they were specialized in the production and secretion of proteins. The pDCs, also known as “natural interferon producing cells”, produce large amounts of type I interferons upon microbial or viral stimulation (44, 45). The pDCs are characterised by high expression of the IL-3 receptor (CD123) and by expression of BDCA-2, a C-type lectin that internalizes antigens for presentation to T-lymphocytes; pDCs also express BDCA-4, a neuronal receptor (46).

DC lifecycle, maturation concepts and migration patterns

Lymphocyte tissue tropism was reported more than 30 years ago (47), and it has been extensively studied in the last decade (48, 49). Do DCs obey the same or similar adhesion “cues” that lymphocytes do, or do they have their own set of “zip codes” in order to reach their target tissues? Knowledge about DC migration control is important and relevant from a therapeutic standpoint: DC-based vaccines e.g. should be able to reach the appropriate tissues/lymph nodes and generate protective immune responses.

DCs sample the peripheral microenvironment and migrate towards secondary lymphoid organs (SLO) where they prime naïve lymphocytes. Thus primed, CD4+

and CD8+

lymphocytes differentiate into activated effector/memory T-lymphocytes imprinted with new homing properties. Early investigations demonstrated DC migration from a tissue to its draining lymph node, where antigen presentation was observed (50).

After lethal irradiation, it was revealed that replacement and repopulation of DCs in the vagina of mice occurred after 12 days (51). Following mesenteric lymphadenectomy and irradiation of rats, DCs appeared after 24-48 hours in the thoracic duct and peaked after three days, which was interpreted as a very rapid turnover of gut DCs (52).

Using bone marrow chimeric mice it was shown that during adult life most LCs would not be replaced by circulating precursors. Hence it is possible that local haematopoietic precursor cells and differentiated LCs, through self-renewal, are contributing to LC steady-state homeostasis.

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After skin injury, however, LCs were replaced by circulating LC-precursors in a CCR2 dependent way (53).

In mice, lymphoid–tissue resident DCs are the most studied whereas little information is available for their human counterparts. The mice spleen is populated by DCs via blood, and splenic mice DCs express MHC class II molecules and CD11c. Two major CD11c+ DCs subsets can be identified; these are CD4+CD8α-CD11b+ which are localized mostly in the marginal zone, and CD8α+CD4-CD11b- localized mostly in the T lymphocyte zone (12, 54). The CD8α- splenic mice DC subset has been shown to present antigens via MHC class II molecules (55) while it was shown that CD8α+ DCs seemed to be specialized in presentation via MHC class I molecules (56).

From studies on mice, it was observed that 5% of the DCs and their progenitor cells in the spleen and LNs were undergoing cell division. Taken together, it is proposed that local proliferation is an important mechanism for lymphoid tissue DC homeostasis. Long-term maintenance on the other hand, is achieved by DCs circulating in the blood (26). Other studies also support the possibility that lymphoid organ DCs are being replenished by blood-borne DCs (57).

Thymic mice DCs are mostly of the CD8α+

phenotype and are localized predominantly in the medulla. Current evidence suggests that a major part of the thymic DC population is generated within the thymus and a minor population is derived from the blood (59). Studies suggest that thymic DCs take part in negative selection of T-lymphocytes (60). Circulating DCs have been shown to enter the thymus and are probably involved in tolerance establishment by collecting antigens in the periphery (61).

Dendritic cells of the mucosa-associated lymphoid tissues (MALT), i.e. the tissue in the Peyers Patches, the lymphoid tissue in the nasal passage, isolated lymphoid follicles in the small intestine, and the appendix of the large intestine are mostly populated by blood-derived DCs (58).

At steady state, mouse pDCs are produced constantly in the bone marrow and migrate via blood to LNs, MALT, and spleen. Human and mouse pDCs have been reported to relocate from the blood via high endothelial venules (HEV) into LNs (62).

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Human pDCs have been observed to circulate in the blood and are found in the fetal bone marrow, thymus, and liver, suggesting that human pDCs develop from HSC in these lymphoid organs (63).

It has been observed that the proliferation rate of mouse pDCs in lymphoid tissues is very low and only 0.3% was in an active cell cycle (64). Taken together, these findings indicate that the lifespan of pDCs, at least in the mouse spleen or LN, is very short and that a constant replenishment from the blood is essential (54).

The paradigm of DC maturation

The main subtypes of DC found in blood under steady state conditions in humans and mice are pDCs and mDCs. They are believed to traffic the blood as DC precursors (pDCs) or as immature DCs (iDCs) and are able to migrate into peripheral tissues where they sense and capture antigens. Once iDCs or pre-DCs encounter microbial or inflammatory stimuli, they undergo a maturation process during which they reduce their antigen capture, induce cytokine production, upregulate co-stimulatory molecules such as CD40, 80, and 86. Mature, immunogenic, DCs are then able to migrate towards secondary LNs where they prime naïve T-lymphocytes (49).

This paradigm, however, has been refined and it has been reported that DCs resident in secondary lymphoid tissue can be both in an immature and a mature state, only a fraction of the secondary lymphoid tissue resident DCs are derived from cells previously resident in peripheral tissues (55, 65). Moreover, stimulation of immature DCs by microbial products and/or inflammatory cytokines has been observed to temporarily enhance endocytosis and antigen processing (66, 67). Migrating DCs, carrying antigens from peripheral tissues, have been shown to induce tolerance besides activating T-lymphocytes (68-70).

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DC migration

Migration of DCs to and from peripheral tissues depends on expression of chemokine receptors and their respective cognate ligands, as well as on different adhesion molecules, such as integrins. Expression of CCR1-CCR7 and CXCR1-CXCR5 by human blood DCs (mDCs and pDCs) has been reported (71). Upon maturation induced by LPS or CD40L, human blood mDCs and moDCs were observed to acquire SLO-tropism, i.e. expressing high levels of CCR7 and CXCR4 (72, 73). The explanation for CCR7 upregulation can be that its ligands, secondary lymphoid tissue chemokine (SLC) and EBI1-ligand chemokine (ELC/MIP3β) are produced in SLOs (74). Interestingly, both CXCR4 and CCR7 are also expressed by naïve T-lymphocytes that could favour co-localization of these cell types (74). However, the importance of CXCR4 upregulation is less clear since its cognate ligands also are produced in non-lymphoid tissues (75). It is suggested that DC maturation results in a coordinated chemokine receptor switch from an inflamed/peripheral tissue tropism to SLO-tropism (73).

Migrating DCs will in order to reach inflamed tissue, adhere to the endothelium and extravasate. Human blood DC (mDCs and pDCs) populations where shown to express the cutaneous lymphocyte associated antigen (CLA), which is an E-selectin ligand, that enable them to roll in post-capillary venules in non-inflamed mice (76). In comparison, demonstrating different interaction abilities, immature mouse blood DCs are able to transmigrate across the resting endothelium in vitro, whereas mature DCs are not (77). In human DCs, DC-SIGN, a cell adhesion receptor, is expressed and non-inflamed endothelial cells express high levels of the intracellular adhesion molecule-2 (ICAM-2). The rolling and subsequent transmigration into peripheral tissues by human mDCs is dependent on the interaction between ICAM-2 and SIGN and the interpretation is that the expression of DC-SIGN regulates mDCs migrating from blood into peripheral tissues (78). Mature mouse DCs have been shown to migrate from peripheral tissue into SLO through afferent lymph vessels, a feature dependent on CCR7 expression (79, 80).

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Danger model

Phenotypical and functional changes from immature DCs to mature/immunogenic DCs, which include a shift in tissue-tropism, are proposed to be the product of so called danger/damage signals (81). Such danger signals are pathogen-associated molecular patterns (PAMPs, e.g., LPS, dsRNA, ssDNA, and flagellin) that signal through Toll-like receptors (TLRs) (81). Recently a new set of danger signals has been recognised, endogenously derived damage signals. An example of a damage signal secreted by macrophages or released by necrotic cells is the high motility group box protein 1 (HMGB1). HMGB1 has been reported to act as a chemoattractant for moDCs, whereas mature moDCs did not respond to HMGB1 (82, 83).

Pattern recognition receptors

Different types of pattern recognition receptors (PRRs), are used by DCs to recognize evolutionary conserved structures i.e. PAMPs. (84). PRRs include

e.g. TLRs, NOD-like receptors, C-type lectin receptors (CLRs), and scavenger receptors (SRs) (85, 86). Other receptors involved in antigen recognition are leukocyte Fc receptors (FcRs) (87). Moreover, both IgG and complement proteins can act as opsonins and bind to FcRs and complement receptors (CR) which enhances internalization and degradation (88).

Toll-like receptors

TLRs induce innate immune activation by detecting highly conserved components of pathogens that are either not expressed by mammalian cells or normally sequestered in intracellular compartments which are inaccessible to the TLRs (89, 90). The importance of the intracellular localization of some TLRs has been discussed in the context of reducing the risk of autoimmunity induction (91).

TLR1, 2, 4, 5, and 6 are expressed on the plasma membrane and mainly recognize bacterial products, whereas TLR3, 7, 8, and 9 are localized in intracellular compartments and are believed to be specialized in viral and nucleic acid detection (85, 91).

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Different TLRs recognize specific PAMPs; bacterial lipoproteins and lipoteichoic acids bind to TLR2, viral dsRNA to TLR3, LPS to TLR4, viral ssRNA to TLR7, and unmethylated bacterial CpG DNA to TLR9 (92). Interestingly, DC-subsets differentially express TLRs, which may demonstrate subset differences in reactivity to microbial antigens. Typically, human mDCs express TLRs1-8 (93), pDCs are reported to express primarily TLR7 and 9 (93). Peripheral mDCs produce proinflammatory cytokines TNF-a, IL-6, and IL-12 upon stimulation with LPS, whereas pDCs produce high amounts of IFNs when stimulated with CpG-ODNs (94). When a TLR-receptor interacts with its specific ligand, antigen uptake by DCs is enhanced, leading to enhanced antigen presentation (66); intracellular signalling will induce production of proinflammatory cytokines and maturation (95, 96). Whether or not TLR-ligation affects phagosome maturation is not established and conflicting results have been reported (97, 98).

Fc receptors

FcRs are receptors that bind the Fc portion of immunoglobulins, promoting antibody dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and antigen presentation (99, 100). FcRs have different specificity for specific Ig isotypes i.e. FcγR binds IgG, FcαR binds IgA, FcεR binds IgE, FcµR binds IgM, and FcδR binds IgD (101). The FcεR is often studied due to its involvement in development of allergic responses (88). Fc receptors are divided into activating (FcγRI (CD64), FcγRIIa (CD32a), FcγRIII (CD16), and FcεRI or inhibitory FcRγIIb (CD32b), receptors (101). Therefore, interaction between a ligand, e.g. immune complexes (ICs), and an Fc-receptor will, dependent on the FcRs engaged, transmit signals either via immunoreceptor tyrosine-based activation (ITAMs) or inhibitory motifs (ITIMs) (101, 102). Ligation of FcyRIIa on human moDCs is reported to induce secretion of inflammatory cytokines and increase T lymphocyte stimulation, however ligation of FcyRIIb did not promote DC maturation (99). Human peripheral blood DCs have been shown to express FcγRII and FcεRI (103, 104).

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C-type lectin receptors

Calcium-dependent lectin receptors (CLRs) typically recognize and bind glycosylated antigens (105). Many CLRs are membrane bound and function on DCs as antigen capture receptors leading to T-lymphocyte presentation (106). It has been observed that CLRs can collaborate with TLRs to induce production of inflammatory cytokines from DCs (107). CLRs have also been shown to recognize glycosylated self-antigens and are proposed to be involved in induction of self-tolerance (108).

Several different CLRs are expressed by DCs, including the mannose receptor (MR), DC-SIGN, DEC-205, BDCA-2, Dectin-1, and Langerin. The MR is constitutively being internalized whereas DEC205 and DC-SIGN are internalized upon ligand binding (105). The MR recognizes mannose and fructose residues and uptake via the MR was shown to enhance presentation of soluble antigens to T-lymphocytes (105). DC-SIGN interacts with different adhesion molecules such as ICAM-2 and ICAM-3, regulating both DC migration and DC-T lymphocyte interactions. Dectin-1 that recognizes β-glucan, which is a major constituent of the fungal cell wall, has been reported to promote inflammatory responses (109). Most CLRs are transmembrane proteins that contain at least one carbohydrate recognition domain (CRD). Lectin structures are diverse, regarding both the number of CRDs and the structural motif found in the cytoplasmic domain of the receptor (105).

Complement receptors

The complement receptors CR3 and CR4 are present on human mDCs and LCs (88). Data regarding expression of CR1 by human DCs, are however ambiguous. It is known that both CR3 and CR4 are involved in uptake of opsonised apoptotic cells and it is reported that opsonised apoptotic cells are targeted to mice DCs (218). The expression and density of complement receptors is maturation dependent (88). Complement proteins can act as chemoattractants and the receptor for the complement protein C1q has been observed to be expressed on moDCs and is able to induce chemoattraction to inflamed tissue, matured human moDCs will however not respond (110).

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Scavenger receptors

Scavenger receptors (SRs) were functionally defined based on their ability to bind and endocytose chemically modified forms of low-density lipoproteins (LDL). Initially most studies were focusing on the SRs role in foam cell formation and atherosclerosis (86). Today, SRs have gained interest as PRRs playing a part in the antimicrobial host defence (111, 112).

SRs are glycoproteins, classified into classes A-H. SRs of class A, B, and E-G are expressed by DCs (111-115). Recognition of Neisseria meningitidis via the class A SR was shown to induce proinflammatory cytokines and that may play a major role in the immune response regarding this pathogen by moDCs (116). The class B SR is reported to be required for the binding and uptake of hepatitis C virus (HCV) by DCs. SR class B mediates HCV trafficking into the MHC class I pathway followed by an efficient cross-presentation by moDCs (112).

Endocytosis

Eukaryotic cells and prokaryotic cells are dependent on uptake of external material; cells endocytose nutrients and growth factors for their intrinsic needs, whereas specialized cell types remove unwanted or excessive materials from the circulation or tissue fluid and thus contribute to tissue homeostasis. Endocytosis may be initiated when a specific receptor on the cell surface binds with high affinity to its recognized ligand in a process called receptor-mediated endocytosis. As a result of the receptor-ligand interaction the plasma membrane in the region carrying the receptor-ligand complex will undergo endocytosis. For example, activation of FcRs occurs after binding such ligands as Ig, soluble and particulate immune complexes and will mediate uptake (101). Other examples of receptors that are able to mediate endocytosis are CRs and CLRs (85, 117). Endocytosis can be divided into two broad categories namely, phagocytosis and pinocytosis (118). In mammals, phagocytosis is conducted primarily by specialized cells such as monocytes, macrophages, neutrophils, and DCs. Phagocytosis is related to the internalization of large particles (>500 nm) e.g. pathogens such as bacteria, large debris as remnants from dead cells, or arterial deposits of fat (119). Pinocytosis on the other hand (or cell drinking) refers to the uptake of fluids and solutes (120). Endocytosis may occur through the following four basic mechanisms:

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Clathrin-mediated endocytosis

Clathrin-mediated endocytosis is a process that most nucleated eukaryotic cells use to concentrate and internalize nutrients, hormones, antigens, growth factors, LDL particles through the LDL-receptor, and iron through the transferrin receptor (121, 122). Internalization is carried out by concentration of high affinity transmembrane receptors and their bound ligands into “coated pits”. Assembly of cytosolic coat proteins forms coated pits, the main assembly unit being clathrin (120). In order to form such clathrin-coated pits (CCPs) under physiological conditions the presences of so-called assembly proteins (APs) is required. There are four structural related AP-complexes that each mediates vesicle formation at distinct sub cellular localizations (123). CCPs are then invaginated into the cytoplasm and pinch off from the plasma membrane to form clathrin coated vesicles (CCVs). The formed vesicles/endosomes are about 150 nm in diameter and once the CCVs have formed they rapidly shed their clathrin coat and a sorting process begins (124). Such endosomes are often called early endosomes and will fuse with one another and with pre-existing sorting endosomes; a process in part regulated by Rab5, early endosome antigen 1 (EEA1), and SNAREs (125). Sorting endosomes consist of a large vacuole with tubular extensions and delivery of cargo to sorting endosomes takes place within 5-10 minutes after formation of CCVs (126). Sorting endosomes will then translocate along tubular formations while becoming more acidic, in a process referred to as maturation. The sorting endosome function is to target molecules to their correct destinations. At present three such destinations are known to exist: the plasma membrane, late endosomes, and the endocytic recycling compartment (ERC) (127). The first step in the endocytic sorting process is the luminal release of the ligands from the receptor due to the decreased pH. Most molecules to be recycled are removed and it is believed that most of the plasma membrane molecules are removed by pinching off narrow-diameter tubules. Narrow diameter tubules bud out from the sorting endosome, and then carry away large fractions of membrane components but relatively little luminal volume (127).

The ERC is mainly a collection of tubular organelles that associate with microtubules, most molecules in the ERC are returned to the plasma membrane (127, 128). The transport machinery of the ERC is not completely understood but two examples of proteins that regulate transport from the ERC are Rab11 and EHD1/Rme1 (129).

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Delivery to other intracellular destinations, such as late endosomes and lysosomes is dependent on microtubules. Late endosomes are formed from sorting endosomes, which is accompanied by a reduction of pH to 5-6.0 (130). As discussed above, DCs express many receptors including CLRs, Fc receptors and TLRs that can be used to recognize and internalize both soluble and particulate antigens or immune complexes in a clathrin-mediated way (131-133). It is debated whether or not clathrin-mediated endocytosis is an important mechanism that DCs utilize. However, it is likely that the binding of ligands to receptors will guide the uptake and subsequent destination of the internalized antigen. And therefore clathrin-mediated endocytosis can very well be one of the mechanisms used by DCs to internalize antigens (131, 133). It is noteworthy that many of the receptors are re-used up to several hundred times by recycling pathways. These recycling pathways are necessary for maintaining a proper composition of various organelles and returning essential molecules to their places of origin e.g. into the cell membrane. To maintain a homeostatic regulation of molecules in different compartments, transport rates and membrane trafficking must be controlled and adjusted in response to cell status and stimulation (127).

Caveolae-mediated endocytosis

Endocytosis can also occur through flask-shaped invaginations, so called caveolae (Latin for little caves), typically of a diameter between 50-100 nm, in the plasma membrane of many cell types (134). Caveolae-mediated endocytosis is probably used in signal transduction (135) oncogenesis (136), uptake of bacterial toxins (137, 138), and also viruses such as the non-enveloped Simian virus 40 (139, 140). Caveolae-mediated endocytosis has also been suggested to be a major route for uptake of RS-virus antigens by moDCs (141). The bilayer of a caveolae micro domain is rich in cholesterol and sphingolipids.

The shape and structural organization of caveolae are effectuated by caveolin, a dimeric protein that inserts a loop into the plasma membrane bilayer and self associates to form a striated caveolin coat on the surface of the membrane invagination (120).

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In addition to caveolin a caveolae also contains the GTPase dynamin, which has been observed to be localized at the neck of the flask-shaped caveolae invagination and has been implicated in pinching off the caveolae vesicle in a way similar to that of clathrin coated vesicle (142, 143).

Macropinocytosis

Macropinocytosis involves membrane ruffling and may be induced in many cell types upon stimulation. Antigen presenting cells such as DCs will upon stimulation trigger prolonged macropinocytic activity that enables the cells to survey their surrounding milieu by sampling large extra cellular volumes (144). Some bacteria have been shown to trigger macropinocytosis ensuring their own uptake (145). Like in phagocytosis, the Rho-family GTPase signalling cascade that triggers the actin-driven formation of membrane protrusion is involved. However, in contrast to the phagocytic protrusions, these will not “zipper up” along a coated ligand, instead they will form and collapse over, and consequently encapsulate, a large volume of the extra cellular milieu (120). Macropinocytosis can thus be described as a form of bulk uptake of fluid and solid cargo by the formation of large and irregular vacuoles, so called macropinosomes of a diameter of 0.5-5 µm (146, 147), and macropinocytosis has been shown to be efficiently used by DCs for antigen uptake (124, 148).

Immature moDCs were reported to use macropinocytosis at a very high (148) and constant rate (149). Upon maturation, the fluid phase uptake was downregulated (148); in another study macropinocytosis was observed to be temporarily upregulated upon encounter with TLR ligands in immature DCs (66). It is reported that immature moDCs use macropinocytosis to internalize and deliver antigens to MHC class II rich compartments that ensures an MHC class II-restricted presentation. DCs have been reported to be able to prevent degradation of antigens either by keeping antigens in neutral so called macropinosomes (150) or by reducing the lysosomal proteolysis for later presentation to T-lymphocytes (151). In order to handle the excess water volumes when internalizing large volumes through macropinocytosis, immature DCs have been shown to express aquaporins (152).

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Phagocytosis

Phagocytosis is an active and highly regulated process. Clustering of phagocytic receptors by ligation of ligands present on the target particle trigger a signalling cascade and if conveyed via ITAMs it will lead to actin polymerization and cytoskeletal alterations and internalization (153). Phagocytosis may be initiated by the interaction between ligands present e.g.

on a microbe or apoptotic cells and a variety of cell surface receptors such as classical Fc receptors, complement and scavenger receptors are used by the cells (154).

Rearrangements of the cytoskeleton lead to formation of protrusions around the particle in a 'zipper-like process' (155). During formation, membrane- and intracellular-components are transported to the forming phagosome (156). The formed phagosome then becomes accessible for interaction with other endocytic compartments and the phagosome matures leading to a modified membrane composition (157). During phagosome maturation a progressive decrease in phagosomal pH to pH 5.5 is achieved and when fusing with lysosomes creating a phagolysosome it will drop even more to around pH 4.5 (67, 97).

Interestingly, although DCs are considered to be specialized phagocytes, in contrast to neutrophils and macrophages they have a much lower degradation and acidification capacity (158). Details about maturation of the phagosome in different subsets of human DCs are not clear. In a recently published study it was demonstrated that active alkalization of the phagosome by the NADPH oxidise NOX2 in DCs took place (158). Lysosomal protease activity is pH dependent and prevented by alkalization. A slow acidification of the phagosomes in DCs will result in partial degradation, which enables antigens to escape total degradation and thereby be loaded onto MHC class I molecules (159). The active alkalization process necessary for prevention of protease activation is, however, a temporary process and after a period of active alkalization due to production of reactive oxygen species (ROS), the production of ROS is diminished or ceases altogether. At this stage the formation of acidic phagosomes starts and lysosomal proteases are subsequently activated. Once the lysosomal proteases are activated, particulate antigens are degraded and loaded onto MHC class II molecules (160).

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DCs express a relatively low number of proteases in their endosomes, including cysteine proteases, aspartate proteases, and asparagine endopeptidase (161, 162). Consequently it has been suggested that endosomal proteolysis in DCs, as opposite to the situation in macrophages and neutrophils, are aimed towards partial degradation of the internalized antigen rather than total degradation (163).

Antigen presentation by dendritic cells

Antigens are basically of two kinds, either endogenously or exogenously derived. In the first case the antigen can be a “self” protein in the form of a tumour specific protein. In the second case the antigens originate from e.g.

bacteria or virus.

As discussed above, DCs being APCs sample antigens in their environment, whereupon antigens are internalized by endocytic mechanisms and distributed and processed within endocytic pathways (173). Peptide-antigens destined for antigen presentation are transported to the cell surface and displayed for presentation to T-lymphocytes on MHC class II molecules (174, 175). A unique property of the DCs is their ability to prime naïve T-lymphocytes (6). Low levels or absence of co-stimulatory signals during antigen presentation are suggested to result in induction of immune tolerance (164). Direct evidence for such ability amongst DCs has been observed (165, 166). In a MLR, DCs stand out in their ability to stimulate T-lymphocytes. Normally a MLR reaction is carried out with equal number of stimulators and responders but in the case of DCs, one DC is sufficient to activate 100-3,000 T-lymphocytes (167). Immature DCs have the ability to endocytose and capture antigens by several mechanisms. These as discussed above, include phagocytosis, (168-170), constitutive and continuous macropinocytosis (148), use of several receptors that mediate endocytosis such as C-type lectin receptors like the MMR (148), Fcγ and Fcε receptors (171) and DEC-205 (172). An efficient uptake of antigens, far more efficient compared to other APCs, has been observed from the DC population (148, 167).

Interestingly though, no one has reported any specific molecule to explain the efficacy of DC binding to the T-lymphocyte receptor. Instead it seems solely related to the quantitative aspect of molecules aiding in the DC-T lymphocyte interaction (167).

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In all nucleated cells intracellular proteins are degraded into peptide fragments in the cytosol by the proteaosome, a large multiprotein enzyme with proteolytic activity. Proteolysis is an ATP-dependent process that starts by ubiquitin conjugation (176). Peptides thus generated by the proteasome are then translocated into the ER by a specialized transporter that is associated with antigen processing (TAP). On the luminal side of the ER, TAP is non-covalently attached to MHC class I molecules via tapasin, a linker protein. Thereby the MHC class I molecules become strategically located at sites where they can receive peptides. Before a peptide is loaded onto a MHC class I molecule, it is often trimmed by the ER resident aminopeptidase (ERAP) enabling it to bind into the peptide-binding cleft. MHC class I molecules carrying peptides are structurally stable and are transferred by means of the Golgi complex and transported to the plasma membrane. Once expressed on the cell surface the MHC class I peptide complex may be recognized by peptide specific CD8+ T-lymphocytes (177).

DCs are also able to present extra cellular components as endogenous antigens via their MHC class I molecules in a processes called cross-priming/presentation (Hereafter called, cross-presentation), (178, 179). Several types of endogenous antigens and intracellular components from other cells such as antigens from tumour cells, virus infected cells or, intracellular bacteria and parasites, as well as immune complexes have been reported to be endocytosed and presented by DCs (180). The mechanism of cross-presentation is currently not fully understood and various models have been suggested (181). Recent evidence suggests that processing of extra cellular antigens to be cross-presented involve fusion of the ER with early phagosomes to form organelles with all the required MHC class I-processing machinery. In this model, phagocytosed antigens are retrotransported out of the phagosome into the cytosol and degraded by nascent proteasomes in a process that may involve the sec 61 complex. Antigens are then transported back into the phagosome via the TAP complex for loading onto MHC class I molecules (182, 183). Interestingly, transfer of proteins to be cross-presented on MHC class I molecules by professional antigen presenting cells has also been observed from adjacent cells via gap junctions (184).

An important function of DCs may also be to take part in the peripheral tolerance mechanism and cross-presentation has been suggested to be involved in the induction of tolerance (185).

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Besides being able to present peptide antigens, DCs also process and present lipid antigens on the cell surface and lipid antigens are presented by CD1 molecules. The human CD1 family consists of at least five members, CD1a-CD1e (186) distantly related to MHC class I molecules that also require β2-microglobulin for stability and subsequent expression on the cell surface for recognition by T-lymphocytes (187).

CD1a-d present exogenous and endogenous lipids, whereas CD1e is reported to be exclusively found intracellularly, and is suggested to play a role in lipid processing and transport to other CD1 proteins (188).

Human LCs have been shown to express CD1a, blood mDCs express CD1c, d and no or low levels of CD1a and b. Interestingly, expression of CD1 by moDCs is highly dependent on the serum used in the culture and differentiation process (189).

Particles, for in vitro study

As discussed in this thesis DCs are important initiators of primary immune responses and stand out in their ability to stimulate T-lymphocytes. As DCs plays a central part in the immune system, attempts have been made to use DCs in immunotherapy studies (190). In studies regarding vaccines, drug delivery, or immunotherapy, several different biomaterials have been used as carriers. The carrier can be bio-degradable polymers (poly lactic-co-glycolic acid)(PLGA)), particle emulsions, immunostimulatory complexes (ISCOMs), liposomes, virus-like particles (VLP), viral/bacterial vectors for DNA delivery (124, 191, 192), or non-degradable particles made from polystyrene, latex, gold, or silica. As an added feature, some of the carriers per se have been shown to have an adjuvant effect (124, 192, 193). A common complication when using nano-particles is the formation of aggregates as discussed by Kalkanidis et al., 2006 (194).

The focus of the papers presented in this thesis has been on early events during

in vitro activation and endocytosis by human peripheral DCs. We have

evaluated if non-degradable zeolite particles can be used as a tool for in vitro

studies of human DCs. Therefore the section below will start by introducing zeolite particles and then move on to particles that resemble zeolite particles.

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Zeolites are synthetic or naturally occurring micro porous crystalline aluminosilicates. Zeolites consist of corner sharing AIO4 and SiO4 tetrahedra that create a three dimensional system of channels and cavities with pore diameters in the range of 2.5 to -7.5 Å. Their chemical composition as well as the size and dimensions of the channel system determine the properties of zeolites. The Si/ Al ratio of zeolites can be varied either during synthesis or post-synthetically, zeolites commonly used have a low Si/ Al ratio and typically a high ion exchange capacity. Reducing the aluminium content of the zeolite reduces the ion exchange capacity and such dealuminated zeolites are thermally stable, hydrophobic, and adsorb proteins efficiently (195, 196). Dealuminated zeolites have been used in a wide range of applications, including removal of detergents in protein purification (197) and of toxic preservatives in pharmaceutical preparations of insulin (198).

Owing to their high adsorption capacity for various biomolecules, dealuminated zeolites have been utilized in the delivery of biomolecules into viable cells (199, 200).

Toxicological studies of a naturally occurring zeolite, clinoptilolite, proved the compound to be non-toxic and safe for use in human and veterinary studies, adjuvant effects have also been observed (193). In comparison with other polymeric micro- and nano-particle based delivery systems to DCs, the advantage of using dealuminated zeolites lies in their high adsorption capacity for various biomolecules.

In order to link biomolecules onto the surface of micro- and nanoparticles, a covalent linkage is generally used to attach the biomolecules. Covalent linkage of a protein or biomolecule is usually achieved after surface modification of the particles to create reactive groups, such as carboxyl or amine groups to which biomolecules can be attached. Non-covalently adsorption of biomolecules by means of hydrophobic forces, electrostatic interactions, van der Waals interactions or hydrogen bonding, in general is less efficient. By the use of dealuminated zeolite particles it is possible to non-covalently attach relatively large amounts of proteins, lipids and other biomolecules as shown in paper II-III.

Adsorption of different types of ligands onto zeolite particles can be made in sequential steps, thus building surfaces resembling protein complexes or cell membranes (lipids) containing a secondary ligand such as LPS, as shown in paper II-IV.

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Several types of polymeric microspheres and nanospheres have been evaluated in endocytose studies using DCs (201, 202). DCs have also been observed to phagocytose particles made from different synthetic materials, including particles of latex, polystyrene, and PLGA (203-205). Uptake of particles is believed to be dependent on various physical properties of the micro particle. In DC uptake studies using polystyrene particles, size was shown to be an important parameter, in that smaller particles, i.e. 40-50 nm, were more efficient in promoting a Th1 response, whereas larger particles (>0.5 µm) rendered a Th2 response (206). It has also been reported that in order to achieve optimal uptake using DCs, particles of a maximal diameter of 0.5 µm should be used. However, DCs also internalize larger particles (>1 µm); uptake of particle of such size is enhanced by introducing a positive surface charge on the particles (207). The surface charge on the particles has also been reported to affect the level of the immune response. Cationic micro particles where efficiently phagocytosed by macrophages and DCs (207, 208), possibly due to the interactions between the negatively charged cell surface and the positive charge on the carrier which might initiate binding and internalization as suggested (207).

Proteins or opsonins on a particle surface have been observed to affect the endocytosing capacity of blood DCs, moDCs, and monocytes. In comparison to BSA coated particles, an enhanced uptake was observed when IgG coated particles were used (208, 209)

Due to differently expressed receptors amongst DC-subsets, a possibility is given to direct the uptake of specific ligand coated particles to distinct DC-subsets. Uptake of particles through specific DC cell surface receptors has been discussed (210) and was also observed by adsorbing different ligands on to the particles used (209).

In summary, to obtain an effective immune response, recognition of the pathogen is pivotal. The mononuclear phagocyte cell system that includes monocytes, macrophages, and DCs is of essential importance to ingest, destroy and also present part of the pathogen to T-lymphocytes in order to activate the adaptive immune system. However, different immune evasion strategies are employed by pathogens including antigenic variability, inhibition of phagolysome formation, and disruption of the phagosome membrane with escape of the pathogen in to the cytoplasm. Several of the immune evasion mechanisms are currently not fully understood and probably involve intrinsic pathogen host interactions.

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A tool in such studies could be a particle of proper size with the ability to adsorb ligands in order to resemble the pathogen in question. Coating micro particles such as zeolite particles with a wide range of different biomolecules could perhaps be such a tool and hopefully also provide further understanding of the mechanisms involved in immune evasion mechanisms. Coating zeolite particles with specific biomolecules/probes could also provide further understanding of mechanisms involved in the sorting of antigens into endocytic compartments.

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RESULTS AND DISCUSSION

DCs are believed to exhibit unique abilities to initiate and modulate innate and adaptive immune responses. DCs are thus widely used in immunotherapy studies. DC based studies, hold clear promises for the development of new approaches for treatment of cancer and other diseases. However, many immunotherapy studies conducted are using mouse DCs or moDCs. With regard to human blood DCs, literature reports are few and fundamental knowledge in terms of recognition, activation, endocytosing, and antigen presenting mechanism is limited. Hence, even if it is a time consuming process to isolate DCs from human peripheral blood and only a few cells are generated, the uniqueness of blood DCs merits the effort. In order to study the early events occurring during recognition and activation of human DCs we have used different TLR-ligands and in combination with endocytosis studies, ligand coated zeolite particles. We were able to adsorb biomolecules on the zeolite particles surfaces resulting in a controlled surface density and we were thus able to direct the uptake of coated zeolite particles to human DCs by the engagement of different receptors. Therefore, we conclude that the application of ligand coated zeolite particles is both a practical and useful tool to gain more understanding about the immunogenic properties and mechanisms of DCs.

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AIMS

The aim of paper I was to study early events during in vitro activation of human peripheral blood DCs by different PAMPs.

The aim of paper II was to investigate the ability to adsorb various biomolecules (LPS, LTA and CpG) on to zeolite particles with the purpose to study receptor-mediated endocytosis.

The aim of paper III was to investigate the endocytic capacity when altering the ligand density on the zeolite particle and to investigate selective, receptor-mediated endocytosis.

The aim of paper IV was to study the endosomal micro milieu after Fc- or TLR-mediated endocytosis by the use of zeolite particles; and whether or not proteins adsorbed onto zeolite particles could be processed and presented by APCs.

PAPER I

Early activation markers of human peripheral dendritic cells

Immature DCs isolated from human peripheral blood by positive selection using magnetic beads, show two distinctive populations after staining with antibodies against CD11c and CD123. The two major populations of DCs in peripheral human blood where further identified and phenotypically characterized as being either CD4+

, CD11+ , CD33+ , MHC II+ , or CD4+ , CD123+ , BDCA2+ , BDCA4+ , MHC II+

. The latter was also found to produce IFN-α upon in vitro stimulation with CpG containing ODN-sequences. The two populations of DCs therefore correspond to myeloid (mDCs) and plasmacytoid (pDCs) DCs respectively. The two DC populations differentially express TLRs important for the activation of DCs; the mDC population expresses TLR-4 while the pDCs express TLR-9 (93). In order to investigate the early activation events in peripheral DCs, the cells were in vitro stimulated with ligands for TLR-4 (LPS) or TLR-9 (CpG) (85).

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In general, several cell surface molecules including CD40 and MHC class II are upregulated upon DC activation (167). In our studies the earliest upregulated co-stimulatory molecule observed was CD40 and it was used as a verification of activation of the DC populations. Activation of peripheral DCs with LPS or CpG also resulted in production of several cytokines into the medium, i.e. TNF-α, IL-6, and high amounts of IL-8.

To analyze the cellular production of IL-8 by the mDC and pDC populations, intra-cellular staining of IL-8 and flow cytometry was performed. Depending on the type of stimuli used, a clear difference in the amount of intracellular accumulated IL-8 was observed from the DC populations.

The rate of production of IL-8 by the two DC populations was investigated by intra-cellular staining of IL-8 after arresting the secretion of IL-8 for 2 hours at various times after induction of in vitro activation. Almost all of the mDCs produced IL-8 after 4 hours of activation whereas less than 50% of the pDC population produced IL-8.

Upregulation of MHC class II molecules is generally considered as an early activation marker of DCs (167). Surprisingly the mDC population showed an upregulation of MHC class II molecules after conditioning in medium at 37 °C that was equal to the upregulation after activation with LPS. Upregulation of MHC-class II on pDCs was seen after stimulation with CpG and in this case no upregulation of MHC class II molecules was observed from the pDCs after conditioning in medium. No significant upregulation of MHC class I was observed by either of the DC populations.

In conclusion activation of peripheral blood DCs with PAMPs induces the production of IL-8 already within two hours of stimulation. Indeed, the production of IL-8 preceded the enhanced expression of the activation marker CD40 in both mDCs and pDCs. Production of IL-8 was the first change in protein expression observed during the activation process of peripheral DCs. Upregulation of MHC class II molecules in response to TLR mediated activation was only observed by the pDC population. The mDC population showed an upregulation of MHC class II molecules without any prior activation, thus care should be taken using increased expression of MHC class II molecules as an early activation marker of peripheral mDCs after in vitro

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PAPER II and III

Receptor-mediated endocytosis of particles by peripheral dendritic cells

Ligand surface density is important for efficient capture of immunoglobulin and phosphatidylcholine coated particles by human peripheral cells

The aim of these studies was to evaluate the capacity of zeolite particles regarding adsorption of hydrophilic, amphiphilic, and hydrophobic biomolecules. Further, it was investigated if the biomolecules retained any biological function once absorbed; i.e. if the adsorbed ligand could be recognized and captured by receptors expressed by human peripheral DCs. We also investigated if capture was dependent of size or on ligand density on the used particle.

As discussed above, human peripheral dendritic cells express an array of receptors needed for recognition and endocytosis of pathogenic structures present in their environment. One family of such pattern recognition receptors is the Toll-like receptors that have a unique expression pattern on the different DC-subsets; this may generate differences in reactivity to pathogenic structures between the subsets (93, 211)

Human DCs subsets differ in the expression pattern of FcγRs; mDCs express both FcγRI and FcγRII (99, 212) whereas pDCs express FcγRII (99, 213). In paper II and III we have showed that zeolite particles have the ability to bind various hydrophilic, amphiphilic, and hydrophobic biomolecules. We were also able to vary the amount of ligand adsorbed. Interestingly, adsorption of all types of ligands used could be made by passive adsorption without any need of covalent linkages. In order to study capture of the coated particles we used fluorescent tagged ovalbumin as a tracer and sequential adsorption of ligands could be performed maintaining the initial amount of adsorbed ligand.

Differently coated zeolite particles were used to investigate receptor-mediated endocytosis by human peripheral DCs and zeolite particles were coated with LTA, LPS, or CpG-ODNs that are reported to engage TLR 2,4 and 9 respectively (94, 95).