Basophils And Or DCS Biology Essay

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Allergic disorders like anaphylaxis, eczema, hay fever and asthma afflict about 25% of the people in developed countries. These allergic disorders are a consequence of persistent or repetitive exposure to allergens, which are mostly common in our environment. People that have allergic disorders develop a strong T helper type 2 (Th2) response (which is part of the adaptive cellular immunity) when in contact with a variety of these allergens like house dust mite, pollen or insect debris [1]. The activated Th2 cells then contact B cells and start to produce immunoglobulin E (IgE) that sensitizes mast cells and basophils by binding to their FceRI receptor. Repeated exposure to the same antigen activates the IgE bound to mast cells and basophils to secrete chemical mediators, cytokines and chemokines that result in the pathological reactions of immediate hypersensitivity [1]. This is called the early phase reaction. Late- and chronic phase reactions result in long-term changes in structure and impaired functioning of the organs [1].

The induction mechanisms of a Th2 response from naïve CD4+ T lymphocytes when exposed to allergens are well known. So far Literature tells us that a naïve T CD4+ lymphocyte requires three signals in order to be activated and differentiate into a Th2 cell: The first signal is an antigen of the allergen in the context of MHC-II. The second signal is a co-stimulatory molecule and the third, last signal that is required for activating Th2 cells, comes from instructive cytokines (especially Il-4) important for Th2 differentiation [1]. IL-4 is required to activate the transcription factors STAT6 and GATA-3 in order to promote Th2 differentiation [2].

The first 2 signals can be provided by a professional antigen presenting cells (APCs), so called Dendritic cells (DCs). They are able to present antigens of an allergen in the context of MHC-II and can give the co-stimulatory signal B7. However, DCs were never found to produce IL-4 that is required to completely induce a Th2 response [3]. Instead, a leukocyte that represents less than 0.5% of the total leukocytes, so called a basophilic granulocyte have been shown to be able to produce Th2-promoting cytokines, including IL-4 [4]. These findings suggest that basophils can function as accessory cells for Th2 differentiation. So far, little was known about the contribution of basophils to the immune responses. But evidence that basophils are involved in the Th2 responses is increasing. It has been found that infection with helminthes (a parasite) strongly induces Th2 cells activation and the proliferation of basophils in the spleens and livers of host mice [5]. This also suggests that basophils might be involved in the induction of Th2 responses.

Strikingly, recent research by Sokol et all [6] claims that basophils function as antigen-presenting cells for an allergen-induced Th2-IgE response. Another recent article by Yoshimoto et all [7] takes it a step further and claims that basophils contribute to Th2-IgE mediated immune response in vivo via IL-4 production and expresses MHC class II complexes in order to present peptides to naïve CD4+ T cells.

These new findings suggest that basophils are key mediators in the Th2-IgE immune response and contribute to allergic disorders. Thus, basophils might be an important therapeutic target cell.

But then a recent article by Hammad et all [3] claims that DCs and not the basophils are necessary and sufficient for the induction of Th2 immunity to inhaled house dust mite allergen. These findings bring up questions whether, basophils or DCs are involved in the induction of a Th2-IgE response and if basophils are a possible therapeutic target or not, for treating allergic disorders. As a result of these conflicting views between the identities of the APCs in Th2-IgE responses, we aim to identify the APC in a mouse model for allergic rhinitis (AR). By identifying the APC in AR we hope to determine whether basophils are a possible therapeutic targets for the treatment of AR. This mouse model for allergic rhinitis closely resembles characteristic effects allergic rhinitis in humans.

Description project

Experiment 1. Can basophils and or DCs activate naïve CD4+ T cells upon grass pollen interaction in vitro?

We want to start our experiments with a so-called pilot study. In this study we want to investigate if basophils and or DCs are able to induce a Th2 response in vitro. Here we will study in vitro if basophils and DCs are able to induce a Th2 response (by functioning as an APC), by bringing basophils or DCs into contact with naïve CD4+ T cells upon grass pollen interaction.

Experiment 2. Can basophils and or DCs induce a Th2-IgE respons in vivo?

In vitro, possible unknown mediators from the body that could contribute to the APC and T cell activation are not present. These mediators do are present in in vivo studies. This could mean that results from experiments in vivo are different from in vitro experiments. Therefore we will perform an experiment in vivo. In this experiment we are questioning whether DCs and or basophils are present on the site of inflammation in trachea/lymph node when stimulated with grass pollen allergens and if they act as APCs. We will also try to answer the same question asked in experiment 1: are the basophils and DCs able to induce a Th2-IgE response? What will be the ratios of these cells inside the lymph nodes and trachea?

Experiment 3. Are DCs depleted mice able to induce a Th2-IgE response?

Our main question is whether basophils or DCs act as APCs to activate naïve CD4+ T cells. Inactivating one of these possible APCs could help us answering this question. If there is a Th2-IgE reponse when stimulated with allergens in the absence of one these APCs, it could be possible that the APC that is absent, is not involved in presenting antigens to naïve CD4+ T cells.

Experiment 4. Are Basophil depleted mice able to induce a Th2-IgE response?

This experiment will contain the same setup as experiment 3, only now we will use a mouse where the basophils are depleted. This is possible by using Mcpt8DTR mice [9]. If there is a Th2-IgE response when stimulated with allergens in the absence of basophils, it could be possible that DCs is involved in presenting antigens and basophils not.

Methods and techniques.

Mice

For our experiments we will use 8 to 12 weeks old female BALB/ c 4get mice. These transgenic mice have an immune system optimized for our experimental demands and contain eGFP IL-4. This is useful for tracking down the IL-4 production in all cells.

For experiment 3 and 4 we will use double transgenic BALB/ c 4get, (CD11c/Mcpt8) DTR mice. These are mouse models that can be depleted from either their DCs or basophils. These mice have an added Diphtheria receptor on either their CD11c or Mcpt8 cell. By injecting these mice with Diphtheria all the CD11c (DC) or Mcpt8 (basophil) cells will be destroyed.

Flowcytometry

This is a technique for counting microscopic particles by using a beam of light and (fluorescent) detectors. Each suspended particle passing through the beam scatters the light and is picked up by the detectors. By analyzing the fluctuations in brightness at each detector, its is possible to derive various types of information of each individual particle.

FACS

For separating cells we will Fluorscence-activated cell sorting (FACS). This method allows us to sort a heterogeneous mixture of cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. Labeling the cells of interest with a dye allows us to separate them from the mixture.

ELISA

In order to determine if there has been a Th2-IgE response in vivo, we will use the ELISA technique. This technique is based on the binding of proteins attached to anti-bodies that specificly target our IgE of interest. When the protein and antibody have bound to the specific allergen, a dye will be activated. Now the allergens are visible and can be measured.

Immunohistochemistry

To determine the presence of specific components inside a tissue, we can use immunohistochemistry. This technique also uses specific anti-bodies that are labeled with a dye to localize components of interest.

Magnetic-activated cell sorting

This method allows the separation of various cell populations depending on their surface antigens (CD molecules). This is done by selecting antibodies with magnetic beads that are specific for the cell of interest. A strong magnetic field allows the cells attached to the beads to be separated.

Description experimental setups

Experiment 1

For this experiment we will use 8 to 12 weeks old female BALB/ c 4get mice. From these mice we will gather bone marrow cells from the femurs of the mice. The obtained bone marrow cells are then differentiated into basophils and DCs as according to the protocol described by Hammed et al [3]. In order to promote basophil survival in vitro, Il-3 is added to the cell culture [3]. Naïve CD4+ T cells will be isolated from the spleen by using magnectic-activated cell sorting. By labeling these naïve CD4+ T cells with CFSE we can keep track of the cell proliferation.

In the cell cultures, the different cell types will be kept on a ratio 5 (T cell) : 1 (APC). These proportions will optimize T cell and APC interactions. The experimental setup exists of 4 groups; each group will be repeated 8 times.

Group

Containing

1

Basophil

CD4+ T cell

Grass pollen

2

Basophil

CD4+ T cell

3

DC

CD4+ T cell

Grass pollen

4

DC

CD4+ T cell

Four days after the start of the experiment, the cell mixtures will be harvested and sorted by FACS. To check if basophils or DCs are expressing the key mediators (Il-4, MHC-II and B7) that are required for CD4+ T cell activation, FACS specific for each of these mediators will be used.

Now to determine whether basophils and or DCs have activated CD4+ T cells, we will look at certain parameters that identify T cell activation:

Because our mice contain eGFP IL-4, we can look at the fluorescent activity if T cells are expressing IL-4 (which is a characteristic of activated CD4+ cells). To quantify the exact amount of IL-4 produced, we will use ELISA specific for IL-4.

Activated T cells will express the specific antigen CD69. By using FACS we can separated the activated T cells from the non-activated T cells. The activated T cells collected this way can be checked for Th1 and Th2 specific markers. Th1 cells will express TBET and produce IFN-y, whereas Th2 cells will express GATA3 and produce IL-5. By doing a PCR on the Th1 and Th2 specific markers and flowcytometry we can unveil the identity of the activated T cells.

By looking at the CFSE labeling of the T cells, we can analyze the cell number, position and division status. These data can also tell us something about the activation of T cells.

Experiment 2

For this experiment we will use BALB/c 4get transgenic mice that contain IL-4 eGFP. The transgenic mice are sensitized with grass pollen intra-tracheal according to the allergic rhinitis model [8]. After immunization the draining lymph nodes and trachea are isolated at day 1, 3, and 7 (including a control).

To examine the Th2-IgE response, the allergen specific IgE titers are measured with ELISA. To determine which cells are present in the lymph nodes and trachea we will use FACS for markers for basophils, DCs and T cells. We will also make an immunohistochemistry for basophils, DCs and T cells to examine exactly where they are located. If there are basophils and DCs present we will look at the relative ratio of these cells and check for IL-4 production, MHC-II and B7 expression by using FACS.

To determine which cells have taken up the allergens, we use fluorescent allergens.

Cells that have taken up fluorescent will now become visible fluorescent allergens inside the cell. Cells that have taken up allergens could be possible APCs.

Experiment 3

In this experiment we want the mice to be DCs depleted. So here we will use the CDc11c-DTR mice. We will sensitize the CD11c-DTR mice intra-tracheal with grass pollen. After immunization we will isolate the lymph node and trachea. First of all we want to be sure that all DCs are depleted and so DCs where not involved in this process. By staining the isolated lymph node and trachea for DCs we determine their presence. Then we want to investigate if a Th2-IgE response has been induced, by measuring IgE titers. This can be achieved by using allergen IgE specific ELISA. Ofcourse we want to know if the possible induced Th2-IgE response has been caused by the basophils. To investigate this, we will look on the site of inflammation if the basophils are present. We will confirm the absence or presence of basophils by using 2 methods: FACS (for Mcpt8) and immunohistochemistry to be sure that we determined the absence or presence of basophils correctly.

Having basophils present on the site of inflammation does not confirm that they induced the Th2-IgE response itself. To determine if they are able to induce a Th2-IgE response themselves we will look at the expression of the three signals that are required for activation of naïve CD4+ T cells: Il-4 production, MHC-II and B7. Using FACS specific for each of these three signals can achieve this.

To confirm that we are dealing with a Th2 response here, we will isolate T cells from lymph node and after 8 days stimulate them again with grass pollen allergens (anti CD3 and CD28). Then we will do PCRs and flowcytometry specifc for the Th1 markers TBET and IFN-y and for Th2 GATA3 and IL-5.

Experiment 4

In this experiment we want the mice to be basophil depleted. So here we will use the Mcpt8DTR mice. We will sensitize the Mcpt8DTR mice intra-tracheal with grass pollen. After immunization we will isolate the lymph node and trachea. First of all we want to be sure that all basophils are depleted and so basophils where not involved in this process. By staining the isolated lymph node and trachea for basophils we determine their presence. Then we want to investigate if a Th2-IgE response has been induced, by measuring IgE titers. This can be achieved by using allergen IgE specific ELISA. Of course we want to know if the possible induced Th2-IgE response has been caused by the DCs. To investigate this, we will look on the site of inflammation if the DCs are present. We will confirm the absence or presence of DCs by using 2 methods: FACS (for CD11c) and immunohistochemistry. Having DCs present on the site of inflammation does not confirm that they induced the Th2-IgE response itself. To determine if they are able to induce a Th2-IgE response themselves we will look at the expression of the three signals that are required for activation of naïve CD4+ T cells: Il-4 production, MHC-II and B7. Using FACS specific for each of these three signals can achieve this.

To confirm that we are dealing with a Th2 response here, we will isolate T cells from lymph node and after 8 days stimulate them again with grass pollen allergens (anti CD3 and CD28). Then we will do PCRs and flowcytometry specifc for the Th1 markers TBET and IFN-y and for Th2 GATA3 and IL-5.

Timetable

Within this project, we will focus on determining whether basophils are possible therapeutic targets for allergic disorders. This will be done by using a setup of four experiments. The first experiment is an in vitro experiment and will take about a year to complete. Here we should have found the answer on if basophils and or DCs are able to activate naïve CD4+ T cells in vitro. The second, third and fourth experiment are in vivo. The second experiment will take about a year should answer the question if basophils and or DCs are able to activate naïve CD4+ T cells in vivo. The third and fourth experiment should also answer the question (or enhance the results from the second experiment) whether basophils and or DCs are able to activate naïve CD4+ T cells in vivo. These two experiments should take about one and a half year. The remaining half year will be used for writing a manuscript that contains the results of the experiments. This manuscript will be submitted to journals in the field of allergy and immunolgy and will altogether result in a PhD thesis.

Quarter

Experiment/ deliverables

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Exp 1.

Exp 2.

Exp 3.

Exp 4.

writing

Literature.

1. S.J. Galli, M. Tsai, M. Piliponsky. The development of allergic inflammation. Nature 454, 445-454.

2. Zheng, W., and R.A. Flavell. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell. 89:587-596. doi:10.1016/S0092-8674(00)80240-8.

3. Inflammatory dendritic cells-not basophils- are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen. H. Hammad, M. Plantinga, K. Deswarte, P. Pouliot, M.A.M. Wilart et al. Journal Exp. Med.

4. Ben-Sasson, S.Z., G. Le Gros, D.H. Conrad, F.D. Finkelman, and W.E.

Paul. 1990. Cross-linking Fc receptors stimulate splenic non-B, non-

T cells to secrete interleukin 4 and other lymphokines. Proc. Natl. Acad.

Sci. USA. 87:1421-1425. doi:10.1073/pnas.87.4.1421

5. Min, B. et al. Basophils produce IL-4 and accumulate in tissues after infection with a Th2-inducing parasite. J. Exp. Med. 200, 507-517 (2004)

6. Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response. C.L. Sokol, N. Chu, S. Yu, S.A. Nish, T.M. Laufer, R. Medzhitov. Immunology 10. 713-721.

7. Basophils contribute to Th2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells. T. Yoshimoto, K. Yasuda, H. Tanaka et all. Immunolgy 10, 706-714.

8. C.T. McCusker. Use of mouse model of allergic rhinitis to study the upper and lower airway link. Curr Opin Allergy Clin Immunol. 2004 Feb:4(1):11-6.

9. T. Wada, K. Ishiwata, H. Koseki et al. Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks. J Clin invest. 2010;120(8):2867-2875.

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