Immunology For Pathogenesis Of Asthma

Bronchoconstriction

Discuss about the Immunology for Pathogenesis of Asthma.

Asthma is a chronic inflammatory disease of lungs and conducting airways. In asthma cells of both immune system like innate and adaptive immune systems work collectively on epithelial cells of airways. The innate immune cells like mast cells, eosinophils, basophils,  macrophages, neutrophils and dendritic cells are responsible for the pathogenesis of asthma. The adaptive immune cells like B cells and T_H2 cells are involved in the pathogenesis of asthma. Due to action of 7 innate and adaptive immune cells following pathological changes occur in asthma patient: bronchial hyper-reactivity (BHR), goblet cell hyperplasia, mucus overproduction, airway wall remodeling and airway narrowing. Most common symptoms of Asthma are frequent episodes of shortness of breath, coughing, sneezing, runny nose, wheezing and chest tightness, pain. There are two types of clinical asthma occur like allergic and non-allergic asthma. Allergic (extrinsic) asthma mainly caused due to inhalation or ingestion of allergens like house dust mite, peanuts, fungal spores, plant pollen and animal dander. In allergic asthma serum immunoglobulin E (IgE) antibodies are present and patients with allergic asthma exhibits positive skin-prick test to the lipoproteins of allergens. Nonallergic (intrinsic) asthma occurs in the late age and generally related with chronic rhinosinusitis, nasal polyps and obesity. Intrinsic asthma is not associated with IgE and exhibits negative skin-prick test. 

T cells, specifically T_H2 cells plays important role in the inflammatory process of asthma, hence it became potential target for the treatment of asthma. There are increased CD4⁺T cells in the airways and T helper 2 (T_H2) cells are predominant in these increased CD4⁺T cells . T_H2 produces immune response by secreting the cytokines IL-4, IL-3, IL-9 and IL-13. These cytokines facilitate IgE production by B cells. IL-5 plays significant role in the eosinophil differentiation in bone marrow. IL-9 attracts and facilitates the differentiation of mast cells. In the early stage of asthma, allergens are detected by the dendritic cells and these allergens are presented by the antigen presenting cells to the T cells. Due to these allergens T cell differentiated in the T_H2 type of cells. T_H2 type of cells produce T_H2 type of response which produces interleukin 4, 5 and 13 and it is associated with stimulation of IgE and eosinophilic response. Under the influence of interleukin-4 (IL-4), T_H2 cells interact with lymphocytes like B cells and these B cells procures antibodies like IgE. IgE circulates in the blood and it binds to the mast cell and basophils through IgE specific receptor like FcεRI (a type of Fc receptor). By binding on the cells these IgE sensitize these cells to the allergens inhaled or ingested. When person gets exposed to the same allergen in the later time, this allergen bind to the IgE which is bound on the surface of the mast cells and basophils. When same allergen molecule binds to the more than one IgE-receptor complex, there is the crosslinking of IgE and Fc receptor. Due to this crosslinking there is the degranulation of mast cells and release of histamine, cytokines, interleukins, leukotrienes and prostaglandins in the surrounding tissues. These inflammatory mediators produces different effects like vasodilation, smooth muscle contraction, mucous hypersecretion and nerve stimulation. Late phase response can often occur in asthma. Late phase response occurs due to migration of lymphocytes, macrophages, eosinophils and in chronic cases neutrophils to the initial site. Cytokines produced form mast cells like necrosis factor-alpha (TNF-alpha), IL-1β, IL-6, IL-8, and IL-13 are responsible persistent pathological changes in asthma.

Airway edema

Bronchoconstriction: One of the important characteristic of the asthmatic respiratory tract is narrowing of the airways and this leads to the improper airflow during respiration. During episodes of acute exacerbations of asthma, airways gets exposed to the stimuli in the form of allergens and irritants and these allergens and irritants produces contraction of bronchial smooth muscle. These allergens produces  bronchoconstriction by virtue of immunoglobulin E (IgE) reliant mediators like histamine, tryptase, leukotrienes, and prostaglandins. There is also non-IgE dependent bronchoconstriction.

Airway edema: As the disease progresses and in the absence of treatment measures for impaired immune mechanism, bronchoconstiction of the airways and consequently resistance to airflow increases. This augmented airflow resistance is mainly due to edema in the airways, mucus hypersecretion from goblet cells and deposition of thickened mucus plugs.

Airway hyperresponsiveness: Airway hyperresponsiveness in asthmatic patients can be measured by evaluating augmented bronchoconstrictor response to muscarinic receptor agonist like methacholine.  It is called as Bronchial challenge test. Factors responsible for the airway hyperresponsiveness are inflammation of airways, impaired neuroregulation and structural changes of airways and lung like hypertrophy and hyperplasia. Degree of airway hyperresponsiveness shows a relationship with methacholine contractile response and severity of asthma.  

Airway remodeling : In asthma structural changes resembling hypertrophy and hyperplasia of the airway smooth muscle occurs. Structural alterations of airways lead to augmented airflow obstruction, airway responsiveness and consequently results in the progressive loss lung function. Currently available medications for asthma are not fully useful for asthma management. Airway remodeling comprises of sub-basement membrane thickening, subepithelial fibrosis, hypertrophy and hyperplasia of airway smooth muscle, blood vessel proliferation and dilation, mucous gland hyperplasia and mucus hypersecretion (1).   

 

Fig.1 Immune mechanism in Asthma

Flow cytometry analysis delivers a rapid qualitative and quantitative explanation of several features of individual cells. With the help of flow cytometry cell size, granularity of cell, features of DNA and RNA, characteristic of cell surface and nuclear markers which are specific for individual cells can be analysed. Bronchoalveolar lavage (BAL) fluid provides very important information for asthma and other lung inflammatory diseases. Flow cytometer analysis of BAL provides profile of different types of white blood cells and corresponding acute and chronic pathological alterations in the lung. In Asthma, composition of BAL in flow cytometry analysis comprised of T_H2 T-cells, eosinophils, dendritic cells and in chronic stages neutrophils. Flow cytometry analysis provides information about surface markers of eosinophils, dendritic cells, basophils, mast cells and neutrophil by using specific antibodies against these surface markers. Commercial antibodies like MHCII, CCR3, CD3, B220 and CD11c are available for the surface markers on T and B cells. By using these antibodies, flow cytometry can be used to differentiate subsets of these cells like T_H1, T_H2, T_H17, T_H22 of T cells. Mainly T_H2 cytokines like IL-4, IL-5, and IL-13 and IgE plays prominent role in asthma pathology. Flow cytometry analysis of BAL provides information about these cytokines and chemokines in association with cluster of differentiation (CD) markers. Frequency of occurrence of these inflammatory cells and cytokines in the BAL of the asthmatic patients depend on the severity of the disease. In severe cases of asthmatic patients there is the manifestation of dual T_H2/T_H17 cells. These inflammatory cells can also measured in the peripheral blood by means of flow cytometry. In the diagnosis of asthma using flow cytometry, these cells can analyzed in the induced sputum of the patient, however it has been observed that there is more variability in the data obtained from the sputum sample. Even though, flow cytometry is not used in routine practice for diagnosis of asthma, it is beneficial in the differential diagnosis of inflammatory lung disease (2, 5).
 

Airway hyperresponsiveness

Fig.2 Flow diagram for Flow Cytometry procedure 

For flow cytometer analysis BAL fluid should be analyzed immediately after collection and cells should be isolated by means of centrifugation. After centrifugation, supernatant should be distributed in small aliquots. Isolated cells should be either cultured with RPMI 1640 with 10% FBS overnight or immediately fixed in 4 % paraformaldehyde for processing in flow cytometer. Cells should be stained with respective antibodies. 

ELISA test is a method of analyzing immune response of body by reaction of immune components and chemicals. Components of ELISA are enzyme which catalyzes biochemical reaction and antigen or antibody which act as immunogenic molecules. Most of the cytokines detected in the asthma patients can be analyzed by ELISA and ELISpot Immunoassays. Cytokines are analyzed by sandwich ELISA.  In sandwich ELISA plate is coated with antibody,  to this sample containing antigen is added, subsequently antigen capture antibody IS added, detecting antibody binds to antigen, enzyme linked secondary antibody is added which binds to detecting antibody, then substrate is added which is converted to detectable form by enzyme. In asthma patient cytokines can be analyzed in BAL, blood and sputum using ELISA method. ELISA has been used utilized in the cytokine estimation in the urine of asthma patients. Commercial ELISA kits are available for the cytokines is asthma patients like IL-4, IL-5, and IL-13, chemokines like CCL11, CXCL10 and CCL17. Markers of the airway remodeling in asthma patients like matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1) and vascular endothelial growth factor can be analyzed in the BAL of asthmatic patient. IgE antibodies in asthma patient can be measured using ELISA (2, 3, 5).            

It has been observed that there is correlation between altered levels of cytokines estimated by means of ELISA and disease severity. There is strong correlation observed between the increased level of IL 4 in BAL of asthma patient and forced expiratory volume 1 (FEV1).With increase in IL4, there is the decrease in FEV1 in patients with asthma. Plasma and BAL TNF-α level estimated using ELISA method are negatively correlated with FEV1. Decreased FEV1 is related to the increased airway wall area thickness and decreased luminal area of airway which are airway remodeling in asthma patients. It reflects airway remodeling in asthma patient and it is related to the cytokine level in BAL of asthma patients estimated by means of ELISA. In non-asthmatic people vital capacity (FVC), residual volume (RV), and residual volume/ total lung capacity (RV/TLC) are higher as compared to the asthmatic patient. These all the parameters are particularly useful in the lung function tests. Impaired functioning of the lung in asthma reflects altered lung function test and this altered lung function test is correlated with the inflammatory and immune cytokines estimated by means by ELISA method. IL-13 estimated using ELISA gives assessment of treatment effect of antiasthmatic medicine like inhaled corticosteroid/Long-acting beta agonists (ICS/LABA). There is correlation between decreased level of IL-13 and increased FEV1 after treatment with ICS/LABA in asthma patients (2).

Airway remodeling

IL-22 can be estimated both in BAL and serum of asthma patients using ELISA method. It has been observed that IL-22 levels are more in severe asthma patients as compared to mild asthma patients and healthy individuals. It reflects IL-22 estimation using ELISA can be applied in differentiating severity of asthma and plan treatment strategy according to the severity of asthma. 

T regulatory cells can be analyzed using flow cytometer. CD4 T regulatory cells plays important role in silencing IL-4 genes. Thus T regulatory cells plays important role in controlling lymphocyte associate airway inflammation. There is increased T regulatory cells through FoxP3 expression or CTLA4 expression, analyzed by means of flow cytometer in patients with moderate to severe asthma (3, 4).      

T_H17 cells have important role in the incidence of steroid-resistant (SR) asthma. T_H17 cells produce IL-17A and IL-22 cytokines. IL-17A and IL-22 cytokines estimated in the peripheral blood mononuclear cell (PBMC) culture using flow cytometer revealed that there is more percentage of IL-17A and IL-22 cytokines in moderate to severe asthmatic patients as compared to nonasthmatic people. There is more percentage of IL-17A and IL-22 cytokine in the steroid resistant (SR) group as compared to the steroid sensitive (SS) group. This is because glucocorticoids didn’t exhibited effect on the IL-17A and IL-22 cytokines. It reflects treatment effect of drugs on cytokines in asthma can be evaluated using flow cytometer. It has been established that there is correlation between the increased IL-17A level and subepithelial fibrosis which is characteristic of airway remodeling in asthma patient. Thus flow cytometer analysis of IL-17A can be directly correlated to the pathological changes in the asthma patients. Moreover, flow cytometer analysis of IL-17A can be applicable to diffentiate between SR and SS (5).       

T_H2 cells have the capability to differentiate further into dual positive T_H2/T_H17 cells. In the BAL of asthma patients there was the increased frequency of T_H2/T_H17 cells analyzed by the flow cytometry (Table 1).

Table 1: Comparison of clinical features of the T_H2/T_H17 low, T_H2 predominant, and  T_H2/T_H17predominant subgroups of asthmatic patients. 

Parameters	TH2/TH17low	TH2predominant	TH2/TH17predominant
IL-4 positive cells	3.0 ± 0.5	22.7 ± 2**	12.6 ± 2
IL-17 positive cells	1.2 ± 0.3	5.9 ± 1.6	6.5 ± 1.5
IL-4/ IL-17 positive cells	1.1 ± 0.3	7.3 ± 1.5	20.4 ± 4*
Total IgE (kIU/L)	111 ± 26	123 ± 26	230 ± 53
FEV1 (%)	79.6 ± 2	73.9 ± 3	59.6 ± 2.7*$
PC20 (mg/mL) for methacholine	4.6 ± 0.5	2.9 ± 0.4#	1.24 ± 0.2*$
Eosinophils/mL of blood	113 ± 9	422 ± 85#	433 ± 57*@
ICS/LABA	15	22	15
SABA	15	22	15

^*P < .05 compared with the T_H2predominant and T_H2/T_H17low subgroups.

^$P < .05 compared with the T_H2predominant subgroup.

^#P < .05 compared with the T_H2/T_H17low subgroup, Mann-Whitney U test.

^@P = .002, all Kruskal Wallis test for multiple comparisons. 

Increased level of IL 4 and IL17 were also detected in the BAL of asthmatic patients, analyzed by ELISA. Increased T_H2/T_H17 cell counts and increased production of IL17 correlated with the increased eosinophil counts and FEV1. These T_H2/T_H17 cells have more steroid resistance as compared to the T_H2 cells alone. Airway obstruction and airway hypersensitivity is also more in T_H2/T_H17 predominant in asthma as compared to the T_H2 predominant asthma. As T_H2/T_H17 cells were identified as prominent reason behind steroid resistance in asthma patients, further research should be directed towards developing T_H2/T_H17 targeted dugs (2). 

References:

Holgate, S.T. 2012. Innate and adaptive immune responses in asthma. Nature Medicne. 18(5): 673-683.

Irvin, C., I. Zafar, J. Good, D. Rollins, C. Christianson, M.M. Gorska, R.J. Martin, and R. Alam. 2014. Increased frequency of dual-positive T_H2/T_H17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma. Allergy. Clin. Immunol. 134:1175-86.

Takahashi, K., K. Hirose, S. Kawashima, Y. Niwa, H. Wakashin, A. Iwata, et al., IL-22 attenuates IL-25 production by lung epithelial cells and inhibits antigen-induced eosinophilic airway inflammation. J. Allergy. Clin. Immunol. 128:1067-76.

Smyth, L.J. C., A. Eustace, U., Kolsum, J. Blaikely, and D. Singh. 2010. Increased Airway T Regulatory Cells in Asthmatic Subjects. Chest, 138(4): 905-912.

Nanzer, A.M., E.S. Chambers, K. Ryanna, D.F. Richards, C. Black, et al., 2013. Enhanced production of IL-17A in patients with severe asthma is inhibited by 1α,25-dihydroxyvitamin D3 in a glucocorticoid-independent fashion. Allergy. Clin. Immunol. 132:297-304.