The list of factors that influence the inflammatory reaction continues to increase. Among these is the introduction of a new class of proteins known as the mitogen activated protein kinase. The MAPK is stated to show much activity and is directly related to the causation of inflammatory reactions. Researches have shown the role of MAPK in activating TNF ? related pathways of inflammation. Only further researches and literature will help us in understanding the extent of the MAPK proteins and its capabilities within the human body. (Wong et al, 2005).
More to the discussion is the role of Interleukin 25 in the MAPK activity, and how it influences the CD 23 expression on the human B cells. IL 25 belongs to the IL 17 cytokine family. (Wong et al, 2005) This protein is secreted by the CD4+ activated memory T cells. It has also shown the capability of inducing IL-4, IL-5, and IL-3. This leads to increase in the concentration of immunoglobulins, including IgE, and can promote conditions such as eosinophilia etc. as mentioned above. (Wong et al, 2005) Therefore, its potential in creating and increasing the frequency of allergic and inflammatory reactions is important to understand.
Moreover, it raises questions about the role of IL 25 in the various pathways such as the MAPK, and how it can influence a patient’s susceptibility to allergies and atopy. Many already claim the role of IL-25 in the conduction of Ig E dependant atopic diseases, and eosinophil-mediated late phase allergic reactions. (Wong et al, 2005) The current research is to study the role of IL-25 in allergic airway inflammation; the relationship between mitogen-activated protein kinases (MAPKs) and asthma; regulation of CD23 antigen expression on B cells.
This is because researches have suggested a very important role of IL-25 in the development of Th type-2 immune response, and therefore can be utilized to suppress inflammatory and allergic reaction pathways. Understanding this area can be very beneficial for patients who suffer from severe allergic conditions and reactions, such as asthmatics and atopic patients. it has been shown that IL-25 via the IL-13 activation is able cause inflammatory changes in the lung tissue, however, the line and methods of how and when is still unclear.
IL-25 however, has shown to cause potent and sustained AHR and inflammation, which is again dependant on the Th type-2 cytokines. This points out to the multiple ways that IL-25 may be able to cause such a dramatic increase in eosinophil concentrations and reactions in a short time. By understanding the mechanisms through which IL-25 can be controlled or blocked in asthmatic patients, the prognosis and outcomes of their illness can be significantly improved. (Sharkhuu et al, 2006) RESEARCH PROBLEM:
IL-25 was first defined and isolated in 2001 with relative importance in controlling the immune system of humans. It operates by activating MAPK, but its specific effect on MAPK is still undetermined. The dose- and time- response of MAPK to IL-25 is still unpredictable and unknown. Thus, it is still not clear how to use IL-25 to produce the required effect. Activated human B lymphocytes express high levels of IL-17 BR (the IL-25 receptor) (Letuve et al, manuscript in preparation) but the action of IL-25 on B-cells is still undefined.
Moreover, CD23 regulate Ig E production in B cells and IL-25 indirectly have effect on IgE production through signaling pathway (MAPKs), so we try to find the effect of IL-25 on CD23 expression on B cells. MATERIALS AND METHODS The time of the study was approximately four months, in which two lines of work was carried out. The first one was the literature accumulation related to our research and the reading of relevant material that will benefit us in the research. The second was a series of experiments conducted by us in order to ascertain the role of IL-25 in the asthmatic inflammatory process.
REAGENTS AND ANTIBODIES USED. The cell cultural supernatants of the G28. 5 hypridoma cell line were used to isolate anti-CD40 antibody or anti-CD 40 of mouse. The company was ATCC, Rockville, MD. The interleukin-25 used in the experiment was taken from (the name of the company). The rest of the material as well as their source is as follows: • Recombinant human IL-4 was taken from R&D systems, Minneapolis, MN. • Phosphor-p38 MAP kinase ie. Thr180/Tyr 182 antibody and Phosphop44/42 MAP kinase (Thr202/Tyr 204) antibody were bought from Cell Signaling Technology (Beverly, MA).
• A FITC-conjugated goat F(ab’)2 anti-rabbite IgG secondary antibody from Jackson ImmunoResearch Laboratories (Burlington ON, Canada). • PE anti-human CD23 antibody was purchased from BioLegend (San Diego, CA). SOURCE OF B CELLS AND CULTURE TECHNIQUES: The experiment utilized human B lymphocytes derived from tonsils of various patients. These patients required removal of their tonsils due to various medical conditions. Prior to the use of the tissue, the patients were asked for consent regarding the use of their tissue for the experiment. The tonsils were then minced.
These tonsils were then put in centrifuge over Ficoll Hypaque (Amersham Pharmacia Biotech, Piscataway, NY, USA) to isolate mononuclear cells from the rest of the tissue. The T and B cells were separated by using the E-rosetting using sheep red blood cells. This was taken from Faculte de Medecine Veterinaire, Universite de Montreal, St-Hyacinthe, QC, Canada. These blood cells were already treated with neuraminidase (Sigma-Aldrich St Louis, MO, USA). Monocytes were depleted by adhering to the plastic. Flow cytometry was used to affirm 98% or greater by flow cytometry.
This technique utilized CD19 staining with less than 2% CD3+T lymphocytes and less than 1% CD14+ monocytes. In carrying out culture experiments, a complete medium was used containing RPMI 1640, 10mg/mL-glutamine, 50 U/mL penicillin, and 50(g/mL streptomycin (Invitrogen, Carlsbad, CA, USA). This was supplemented with 10% FCS (HyClone, Logan, UT, USA) and B cells were suspended in it. RAMOS and U266 B cells were taken from ATCC(Rockland, MD, USA) which were also kept in the RPMI medium. Generally, the B cells (1? 10 raised to power 6) were incubated for 24 hours with or without 1(g/mL mouse anti-human CD 40 antibodies (Abs).
The CD40 antibodies were purified with G28. 5 hybridoma cell supernatant along with 200 U/mL rhIL-4 (R&D Systems, Minneapolic, MN, USA) For specific indications in the experiment, the incubation time of the B-cells was increased. EFFECT OF INTERLEUKIN-25 ON MAPKs METHODOLOGY The following steps were taken. 1X Phosphate Buffered Saline or PBS was used to wash the cell cultures for three times. Centrifugation and aspirate supernatant were used to collect the cells. These cells were then rested in 1mL 1 X PBS and resuspended in a 1 hour incubation time.
IL-25 was added at specific amounts into each well at certain time intervals. STAINING PROCEDURE FOR INTRACELLULAR p44/p42 AND p38 AND FLOW CYTOMETRY PROCEDURE The cells were washed using IX PBS. At appropriate dilution, the primary antibody was added to the assay tubes. These were then incubated at room temperature for 30 minutes. The cells were then resuspended in FITC-conjugated goat F(ab’)2 anti-rabbite IgG secondary antibody. These were first diluted in IX PBS as per manufacturer’s recommendation, and were incubated in the dark for 15 minutes at the room temperature.
Rinsing was carried out in the same manner in 1X PBS by centrifugation. The cells were then resuspended in 400uL 1X PBS and were analyzed on flow cytometer. CD 23 ANTIGEN CELL STAINING PROCEDURE. The cells were washed by using 1X PBS. Then 10uL of CD 23 was introduced in each well, which was then incubated for 30 minutes at 4 degrees celcius. The cells were then again rinsed with the 1X PBS and were resuspended in 200uL 1X PBS. These were then analyzed on the flow cytometer. 4. resuspend cells in 400uL 1X PBS and analyze on flow cytometer.