ABSTRACT: INTRODUCTION: Eosinophils mainly handle the inflammatory and allergic responses of the body, and can be in increased levels in patients with asthma or allergies etc. Many molecules are responsible for the mediation of these functions. IL-3, IL4, IL-5 and IL 13 are some of the most commonly known mediators of inflammation. Other factors that have come to light recently include MAPKs, CD 23 and IL-25. These three factors have shown significant relationship in mediating allergic reactions in humans. IL-25 has shown a very high increase in the inflammatory reactions, which result in eosinophilia and increased cellular migration.
MATERIALS AND METHODS: The aim of the study was • To study the role of IL-25 in allergic airway inflammation. • Relationship between MAPK in the process of asthma • Regulation of CD 23 antigen expression on B cells. For this purpose, four works of research was conducted. After isolating the B cells from human tonsils, other cell lines, the RAMOS and U266 were obtained from ATCC and maintained in complete RPMI medium. B cells were stimulated via the IL-4 and anti CD-40 abs for 24 hours and IL-25 was added later on. The flow cytometry was used to measure the MAPKs activation using phosphor specific antibodies to p38 and p44/42.
The RAMOS cell line was used for the following days to analyze CD 23 and its signaling pathway. RESULTS: The results were observed after 1minute for p44/42 and after 5 minutes for p38. The concentrations taken for IL-25 were 50ng/ml, 100ng/ml, 150ng/ml and 200ng/ml respectively. For MAPKs we found that a significant increase in the level of phosphorated p44/42 was observed when B cells were stimulated with PDB, but there was no change for IL-25 at 100ng/ml stimulation. No phosphorated p38 was detected while a slight but notable increase was detected for phosphorated p44/42 in the first minute.
Generally there was no phosphoration for both p44/42 and p38 in dose response experiment. For CD 23 we found that the baseline in the non-stimulated RAMOs cells was quite high, so the exact trend of CD 23 expression could not be identified. There was a great increase in the CD 23 expression level after the B cells were stimulated. After the addition of IL-25, the amount of CD 23 was increased even further whereas the dose response of IL-25 seems not to effectively influence the CD 23 expression level. CONCLUSIONS: FOR MAPK EXPERIMENT:
The MAPK analysis was not successful, and therefore, a modified technique will b required for the future in order to get better results. FOR CD 23 EXPERIMENTS: For CD 23 expression analysis, the positive control was set up successfully. By this we mean that the CD 23 levels were increased when stimulated by IL-4 and anti-CD 40 antibody, however, the concentration of IL-25 seems not to affect the CD 23 expression. For the future a repeat experiment is required for CD 23 incubation at both 42 and 72 hrs with a small amount of IL-25. The use of ELISA kit in measuring the level of soluble CD 23 in the supernatant will be useful.
INTERLEUKIN 25, MITOGEN ACTIVATED PROTEIN KINASES (MAPKS) AND CD23 ANTIGEN EXPRESSION ON HUMAN B CELLS RESULTS Initially about 100ng/ml of IL-25 was used to stimulate the signaling pathway in the stimulated fresh B cells while flow cytrometry was used to measure the MAPKs activation using phosphor specific antibodies to p38 and p44/42. From Figure 1, 2, No phosphorated p38 was detected while a slight but insignificant increase was detected for phosphorated p44/42. For the second trial, the same concentration of IL-25 was used but a positive control was set up by using 10-8M PDB to stimulate MAPKs activation.
Although a significant increase in the level of phophorated p44/42 for positive control was observed (Figure 3), there was no change for IL-25 stimulation (Figure 4 and 5). For the following days, the RAMOS cell line was used to analyze the signaling pathway. Slight phosphoration was detected only for the first minute in the time-response of P44/42 to IL-25 experiment (Figure 6). The results showed that the concentration level of the second antibody was quite high and needs to be adjusted by series titration.
In dose-response experiments, there was no increase detected in both p44/42 and p38 (Figure 7 and 8). CD23 expression level analysis As the signaling trials proved unsuccessful, CD23 was analysis to find out the effect of IL-25 in stimulated B cells. Compared with the non-stimulated cells, the level of CD23 expression in stimulated Ramos cells decreased, and as the concentration of IL-25 increased from 100 to 300ng/ml, the CD23 expression level decreased successively (Figure 9). However, the exact trend of CD23 expression was difficult to identify due to the high baseline in non-stimulated RAMOS cells.
When fresh B cells were used, it was found that there was great increase in CD23 expression level after B cells were stimulated by IL-4 and anti-CD40 antibody (Figure 10). After the addition of IL-25, the amount of CD23 was increased even further whereas the dose-response of IL-25 seems not to effectively influence the CD23 expression level. Repeating the experiments produced consistent results. A dilute concentration of IL-25 was used as previous experiments have shown that the concentration of IL-25 has had minimal effect on the level of CD23 expression (Figure 11).
The U266 was used for negative control and the results exhibited the lack of significant effect in comparison to both the non-stimulated and stimulated U266. our findings are consistent with the many experiments that were carried out in the same area in the past. The results sow that fresh B cells with IL-4 and anti- CD23 cause a significant increase in the expression of CD 23. The level of CD23 expression increased even further with the addition of IL-25; however, when the concentration of IL-25 reached 150 ng/ml, the level of CD23 expression decreased a little for both incubation periods.
It is unjustified to conclude that 100 ng/ml of IL-25 is the concentration which maximizes CD23 expression, for the results are not consistent with previous findings. A repetition of the experiment is, therefore, necessary. The procedure was modified in the 72 hour incubation, where by IL-4 was added along with CD 40 antibody and IL-25 together in the first day. After 72 hours, the amount of CD 23 was calculated. The results showed that the CD 23 expression level was similar to the older procedure, which confirmed the accuracy of the original experiment.
However the 72 hour incubation displayed a plateau when compared to the 48 hour incubation period. The use of an ELISA kit in measuring the level of soluble CD23 in the supernatant and an in-depth exploration of CD23 regulation from an IL-25 inclusion are suggestions for future research. DISCUSSION IL-17 B receptor, also known as IL_17 Rh1, IL-17ER, and EVI 27, is a 502 amino acid Type I membrane protein with a 17 aa signal peptide. (R&D systems, 2005) It has a 275 aa extracellular domain, a 21 aa transmembrane domain and a 189 aa cytoplasmic tail. By alternative splicing, a secreted variant of IL-17BR can be obtained.
(R&D systems, 2005) This receptor is to be found both in primary B cells and cell lines and utilizes NF? B for signal transmission. The receptor is highly sensitive from signals given by Th2 cells. IL 17-E has been shown to influence the B cells very much and how they function. This has been especially seen in vivo and in the presence of T cell dependant humoral responses. This mechanism of T cell influencing the B cells is still some what misunderstood. (Letuve et al. )There is even lesser information regarding the role of IL-17 E in various human pathologies.
What is known that IL-17E presents itself prominently in patients who suffer from asthma. Other cells on which IL-17 BR can cause its influence includes the fibroblasts and smooth muscle cells. Researchers propose that IL-17E actually works by stimulating the fibroblasts, which in turn leads to the recruitment of the eosinophils and release of the inflammatory mediators such as IAM-1. Again, dendritic and macrophage cells may also come under the influence fo IL-17E. (Letuve et al. ) IL-17BR still remains a controversial issue in the case of B cells, where its presence and expression remain the topic of debate.
The results of analysis of messenger transcripts encoding the receptors in the murine and B cells are very inconsistent. (Letuve et al. ) These transcriptors were found to be completely absent from tissues such as the lymph nodes and splenic tissues in the humans. The inconsistent levels of IL-BR E may be due to the stage and phase of the B cells, which may show low levels of expression and mRNAs during resting phases. This has been supported from studies in which activation of the isolated tonsillar B cells and expression of IL17B E was only achieved through introduction of CD40 ligation in the presence of IL-4.various conditions of B cells including Burkitt’s lymphoma have also shown such similar patterns. (Letuve et al. )
However what has been proved in research is the presence of IL17-BR E expression once the B cells were activated. Therefore, increased B lymphocyte activation is the cause of increased IL-17 BE expression on the cells. This line of thought was followed by experiments, which showed that B cell lines were highly dependant on the presence of CD40 for the activation of the B cells. CD40 for its activity also required the presence of factors such as IL-4 and IL-13 in the process.
Experiments have shown no such role of the factors IFN-? or IL-12. (Letuve et al. ) the preference of presentation and factors is also evident on many scales. Th2 cells may prefer the expression of IL17-BR on the eosinophils and other antigen presenting cells. smooth muscles in the airways however, may be uninfluenced by the IL-4 and IL-13 in Th2 conditions “suggesting that cell specific signaling involved in modulation of receptor’s expression” (Letuve et al. ) Many factors may increase or decrease the expression of these factors.
For example, CD40 induced IL17-B R levels may be decreased if exprosure to IFN? or IL-12 took place. IL-17BR expression is very reduced when exposure to corticosteroid takes place. IFN-? and dexamethasone have been found to downregulate and inhibit the expression of IL-17 BR in the airway smooth muscles, proving the IL-17BE is only responsive and active in the presence of Th2 immune responses and has a very reduced role in the Th1 immune mechanisms. (Letuve et al. )As mentioned, CD40 requires the mediation of the NF-? B for its activation.
In B cells, it utilizes the TRAF2 signalling instead of the TRAF6, which is indicative of the synergistic effect of CD40 ligation and IL-17E at the point of NF-? B. IL-17E however, demonstrates a moderate increase in the B cell proliferation, which may be enhanced by CD40. this suggests that induction is not the main component and function of the IL-17. (Letuve et al. )? The point of dispute lies in the overexpression of the IL-17E but no significant proliferation of the cells or the IgE production. Recent studies however, do suggest that IL-17 E may be responsible for the expression of other immunoglobulin types.
(Letuve et al. This brings to mind a possibility that perhaps the B cells are the source of IL-17 E in the first place and the exogenous expression and introduction is responsible for the endogenous cytokine levels. The levels of the receptors IL-17BR are however, highly sensitive to the inflammatory environment. IL-17BR is reported to e the most strongly induced genes in B cells activation by CD40 ligation and IL-4. current researches point to the potential role of corticosteroid therapy in IL-17 BR regulation. (Letuve et al.).
A small discussion of the IgD is important in understanding various modalities that affect the B cell functioning and acitivity. IgD in the human body may be found in two forms. The first form, the serum IgD is found in very small quantities in the body. (Levan-Petit et al, 1999) The bound IgD form is more prevalent and is found B cell antigen receptors, forming a major part of these. Both IgM and IgD are coexpressed on the surface of the B lymphocytes. The IgD has been found to be more efficient than IgM when comparing signaling routes.
IgD induces tyrosine phosphorylation, which is more efficient when comparing the same mechanism carried out by IgM molecules. (Levan-Petit et al, 1999) The expression of the Ig molecules from the B cells is dependant on many variables including their cell line and development. Here the role of CD 40 assumes shape. Interactions between CD 40 on B cells and its ligand CD40L on activated T cells is claimed critical for the induction of the various Ig isotypes. This selection of various Ig types by particular cell is dependant on the direction taken by the cytokines.
CD40 when stimulates the IL-4 induces the formation of the IgE, IgG1, IgG3 and IgG4 switching and secretion. Other factors also influence the production of one or the other type of Igs. The mechanism with which the IgD production is carried out remains largely unknown till now. Researches demonstrate that “stimulation of PBMC by triggering the CD40 pathway with an anti-CD40 mAb in the presence of the Th2 cytokines IL-4 or IL-10 significantly enhances the production of IgD in vitro in comparison with unstimulated cells or cells stimulated with IL-4, IL-10 or CD40 mAb alone.
In comparison with unstimulated cultures, IgD levels were significantly higher after activation of tonsil mononuclear cells by CD40 mAb and IL-10. Interestingly, we detected IgD earlier in cultures of tonsil mononuclear cells than of PBMC. The presence of differentiated B cells and IgD plasma cells in tonsil (23) could account for this phenomenon. Measurements of IgM and IgE levels performed as positive controls yielded the expected result”. (Levan-Petit et al, 1999) UNDERSTANDING THE RELATION BETWEEN THE IL-25 AND IL-17E Although the IL-25 belongs to the family of the IL-17, there are some very prominent differences that IL-25 exhibits.
It is identified as the fifth member of the IL-17 cytokine family. (Maezawa et al, 2006) IL-25 belongs to the cystine knot family, and therefore requires the correct structural formation for its activity in the biological systems. (Maezawa et al, 2006) These differences have been largely discovered via various in vivo and in vitro studies. The homology with other IL-17 members at amino acid level is only 18%. (Maezawa et al, 2006) cytokines resembling IL-17 include IL-17B and IL-17, which can also promote inflammation and hemopoiesis.
(Hurst et al, 2002) These three have very similar activities. IL-17 causes the production of cytokins and inflammatory mediators, which include IL-6, IL_8 and TNF-?. (Fort et al, 2001) The differences in the various proteins of the IL-17 family lie in the differences of length and structure of the N- and c-terminal regions and in the number of cystine residues. (Sharkhuu et al, 2006) Other reasons for the varying activity of these proteins are due to different receptor complexes, downstream intracellular signaling pathways and expression profiles in various organs.
Again, these cytokines may be regulated by different sets of immune and inflammatory responses for each particular IL-17 protein. (Fallon et al, 2006)These activities are mainly initiated by the stimulation of the monocyte line. These actions are similar despite the difference in the sources of the release of these proteins, and cause neutrophil infiltration in the area. (Fort et al, 2001) IL-17 is mainly present in the CD4+ of the activated kind, and their genetic expression can be found in these cells. (Falon et al, 2004)This gene expression cannot be found in whole tissues.
IL-17 can cause many tissue changes to occur upon their expression. These include splenomegaly as there is migration of the progenitor cells of the bone marrow to the splenic tissue. The expression of neutrophils also increases. If the levels of IL-17 increase in the body,there is infiltration of neutrophils in various tissues, including the lungs. by inducing various cytokines such as IL-1? , IL-6, GRO? and TNF-? , IL-17 can cause granulopoeisis and inflammation. These expressions are very different from the IL-25, which are expressive in the Th2 cells and create very different reactions in the body.
(Fort et al, 2001) IL-17 levels have been found to be elevated in rheumatoid arthritis, asthma, multiple sclerosis, psoriasis and transplant rejection. (Hurst et al, 2002) However, some points of similarities do exist. For example, the levels of both the IL-25 and IL-17E are elevated in patients who have allergic conditions such as asthma. (Falon et al, 2004) These quantities have been found to be considerably higher than the controls that have been used in the various studies. The production of the chemokines by the IL-25 cause many changes in the tissue structures of the organs affected.
In fact these changes may be so marked that they may start to border on the pathological side. (Wong et al, 2005) IL-17A for example can up-regulate various factors such as the TNF-? , IL-1? , IL-6, IL-8, monocyte chemo-attractant protein or MCP-1 and granulocyte-macrophage colony stimulating factor. Other molecules that are up regulated include intracellular adhesion molecule or ICAM-1 and promotion of CD4+ T-helper 1 lymphocyte or Th Type 1. (Sharkhuu et al, 2006) The production of IL-25 from the various cell modalities has been studied time and again. PCR methods have been used to study gene expression of IL-25.
The best way to study the changes that take place due to IL-25 is by studying mouse models. (Sharkhuu et al, 2006)This is because IL-25 of both man and mouse is 80% similar. (Sharkhuu et al, 2006) The T cell population has been found to be less responsive in producing IL-25 than other cell populations. Experiments have shown that T cells that have been stimulated in the presence of 1 to 3 weeks in the presence of IL-4 and IL-12mAb showed any expression of IL-25. These expressions were only seen in the Th2 polarized T cells, but showed no activity or presence in the fresh CD4+ cells.
Experiments with mice have shown the largest production levels in the GIT and uterus. Researchers state that this may be due to the high quantities of T cells in these organs, and the activated Th2 cells may be ones causing increased expression of IL25. Many other white cell populations are being studied about their expressive capability of the IL-25. (Fort et al, 2001) The in vitro activity of the IL-2 is very remarkable, and this protein alone has the capability of activating almost the entire cells of the white cell lineage.
(Wong et al, 2005) For example, the activation of cytokine can activate the Th2 cells. The CXC chemokine IL-8 is activated for the neutrophils. The CC chemokine MCP-1 is released for monocytes; macrophages, eosinophils and Th2 cells, and chemokine MIP-1? for monocytes and natural killer cells. IL-25 also can cause neutrophil infiltration. This correlates with the production of IL-8 from the eosinophils, again mediated by IL-25. IL-25 can therefore act simultaneously on all cells at various levels and can cause inter activation of one cell type by the other.
(Wong et al, 2005) It has also been shown that IL-25 causes induction of IL-8 in the human renal carcinoma cell line. (Maezawa et al, 2006) The role of IL-25 may not stop here alone, for each type of cell line when activated will carry out certain activities of its own. For example, eosinophils up regulate themselves as their cytokines get activated by increasing their chemokine receptors. This means that the up regulated cells will hold the capability to increasing their activity in more aggressive fashion. (Wong et al, 2005).
Mice studies have shown a corresponding increase in the eosinophil count as the exposure to IL-25 increases. Even 10? g of IL-25 for 10 days caused significant changes in the eosinophil count within mice injected with it. For example, splenomegaly was noted and the cells that were increased comprised only of eosinophils and not neutrophils. This is a very good example of selective effects of IL-25 on the eosinophil population. Such number of cells within the spleen was also increased, along with a 20-fold increase in the myeloid colony formatting cells or CFU-c.
This particular finding suggests that IL-25 may also be influencing the progenitor cells of the blood cell line, which migrate to the area, and increase their expression in the blood. Marked eosinophilia was reported in such mice, which was seen within 4 days of the injections of IL-25, reaching a peak on the 7th day of injecting. Again, the blood films only showed an increase in the eosinophil count and not the neutrophils count. With the increase in the production of eosinophils came the rise of these cells in the blood, which again was manifested as eosinophilia. (Fort et al, 2001)
Therefore we can conclude that IL-25 is elevated in allergic inflammation. IL-25 has been found to synergistically induce Th2 cytokines release which includes IL-4, IL-5 and IL-10, inflammatory cytokine IL-6 Th1 related chemokines CXCL 9 and CXCL 10 and chemokine CCL5. (Wong, Li and Lam, 2007) These chemokines and cytokines have been established to play important pathological roles in the progression of allergic diseases. Studies have shown that eosinophils can upregulate their chemokine receptors upon cytokin activation and use them during the progression of allergic inflammation for eosinophilic migration and activation.
Studies support the role of p 38 MAPK and NF-? B in the TNF mediated release of eotaxin from the eosinophils. Intracellular mechanisms also support activation of p36 MAPK, JNK and NF-? B via the IL-25. other experiments show that the production of IL-8, MIP1? , MCP 1 and Th2 cytokine IL-6 from eosinophils can induce or provoke the Th2 response. (Wong et al, 2005) the IL-17 class is known to activate the three classes of the MAPKs, including the ERK1 and ERK2, JNK and p38. in bronchial cells this activation leads to the production of IL-6 and CXC chemokine IL-8 in vitro.
(Kolls et al, 2002) This is the mechanism thought to hel in the recruitment of neutrophils into the lung tissue. The use of neutroalization studies and mice with homozygous deletion of the IL-17 receptor has decreased the recruitment of neutrophils in the lung in response to a challenge from a gram negative organism. (Kolls et al, 2002) This supports the tyhoery that IL-17 may be responsible for innate immune response to the gram negative bacteria. In such conditions the CD 4+ and CD8+ can also cause the production of the IL-17 and cause antigen induced adaptive immune responses. (Kolls et al, 2002)
researches carried out by Wong et al in 2005 suggest that “IL-25–induced release of MCP-1, MIP-1_, IL-8, and IL-6 from eosinophils is mediated by the combined activation of MAPK and NF-_B pathways, thereby providing new clues for the elucidation of immunopathologic mechanisms of Th2- and eosinophil-mediated allergic inflammation in pulmonary disorders. Further investigations are required for other potential intracellular signaling pathways (e. g. , Janus-activated kinase [JAK]–signal transducer and activator of transcription [STAT]) for the regulation of the release of chemokines and cytokines.
In view of recent advances in the application of p38 MAPK and NF-_B inhibitors as potential anti-inflammatory agents in asthma (33, 34), our study of IL-25 on eosinophil activation should provide new insights on the development ofnovel treatment for allergic disorders. ” (Wong et al, 2005) CD 23 has been established to have a very important role in the varius immunological reactions. Both the transmembrane form and the soluble forms if undergo pathology, may exhibit certain pathological conditions.
This is independent on the expression quantity of either the soluble or the transmembrane form of the CD23, as demonstrated on experimental mice. The researchers of the study claimed that neither mCD23 or sCD23 overexpression caused any significant changes in the maturation of the lymphoid cells. the IgE and the IgG1 levels were not significantly increased or altered. (Texido et al, 1994) The effects of CD 23 in various clinical conditions regarding B cells is beginning to give some idea of the mechanisms by which it influences the B cell population.
It has been found that sCD 23 is elevated in patients who suffer from B cell chronic lymphocytic leukemia. The sCD23 is now being evaluated to help in understanding and predicting prognosis and survival rates of patients with CLL. sCD 23 levels are now considered as a valuable tool in diagnosing the survival and prognosis of patients with CLL. This method can be very helpful in predicting those patients in whom the disease may progress and may help in treating them accordingly. (Sarfati et al, 1996) CD 23 is also considered a very important regulator of the IgE.
It has been implication in cellular adhesion antigen presentation as a growth differentiation factor for human B, T and plasma cells and as a signal transduction molecule. Initially it was considered that CD 23 also acted as a growth factor for the B and T cells however the current research and studies have not found any proof regarding this. (Yu et al, 1994) Again CD 23 has been found to be non essential for class switch or for plasma cell differentiation. It is also not important for the clearance of IgE from the serum. Researchers claim that CD 23 may function through negative feedback control of the IgE.
In this regard the role of IL-4 is essential for class switching to IgE and also is a strong inducer for CD 23 as well. CD 23 is also not considered as sufficient to interfere with antigen specific IgE responses. Yu et al, (1994) claim that “binding of IgE containing immune complexes to CD 23 renders the B cell unresponsive to further stimulation or directly suppresses IgE synthesis”. (Yu et al, 1994) This is supported by evidence that IgE containing immune complexes and anti CD 23 antibodies suppress human B cell proliferation and IgE production in vitro.
Another supporting fact in this regard is that anti CD 23 antibody treatment of rats inhibits a specific IgE immune response. (Yu et al , 1994) IL-25 researches show that the production of IL-25 in the lung can result in expression of all signs that are typical to Th2 diseases. These include infiltrate, cytokine production, tissue reorganization, mucus secretion, and air hyperreactivity. (Hurst et al, 2002) The studies also show that while IL-25 can amplify the type II mediated allergic reactions, it does not induce allergic inflammation in vivo.
(Tamachi et al, 2006) IL-25 is mostly produced at the site of inflammation and inhibits antigen induced eosinophil and CD4+ T cell recruitment in to the ariwasy by sIL-25R. IL-25 however is dependant on the presence of CD4+ and Stat6 expression in order to enhance its activity. The IL-25 mRNA can also be increased in many other conditions which include fungal and helminthic infections. (Tamachi et al, 2006) The endogenously produced IL-25 is claimed to be involved in antigen inducing Th2 type immune responses in the airways.
IL-25 requires the presence of the antigen as well to create its effect and simple introduiton of IL-25 without the presence of the antigen challenge will not lead to any respoinse. This finding is in contrast to many studies that state that introduction of IL-25 alone can induce Th2 cytokine production and inflammation and eosinophil infiltration in the lungs. Which is true needs to be evaluated using more research. (Tamachi et al, 2006) CONCLUSIONS: FOR MAPK EXPERIMENT: The MAPK analysis was not successful, and therefore, a modified technique will b required for the future in order to get better results.
FOR CD 23 EXPERIMENTS: For CD 23 expression analysis, the positive control was set up successfully. By this we mean that the CD 23 levels were increased when stimulated by IL-4 and anti-CD 40 antibody, however, the concentration of IL-25 seems not to affect the CD 23 expression. For the future a repeat experiment is required for CD 23 incubation at both 42 and 72 hrs with a small amount of IL-25. The use of ELISA kit in measuring the level of soluble CD 23 in the supernatant will be useful. Basically the signaling pathway analysis was fail, there was no significant results obtained from fresh B cells.
So we move the analysis CD23 expression, which is one kind consequence of signaling pathway. We only set up positive control that CD23 expression level has been great increase on human B cells stimulated by IL-4 and anti-CD40, but the effect IL-25 for CD23 expression cannot determine based on this project.
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