The roles of basophils in mediating the immune responses

Mechanisms underlying basophil activation

Basophil activation is the prerequisite for exerting basophil biological functions. Most studies evaluating the roles of basophils have focused on IgE-mediated hypersensitivity reactions, and basophils have been shown to be activated in response to multiple signaling pathways. Activated basophils can produce various effector molecules (including histamine and leukotrienes), chemokines (IL-4, IL-5, IL-13, IL-6, and tumor necrosis factor [TNF]-α), and cell membrane markers (CD203c and CD63). ¹⁰ The classical activation of basophils occurs when a foreign antigen and IgE form an immune complex, which then binds to the high-affinity IgE receptor FceR1. This process triggers the activation of basophils, which release various inflammatory mediators and participate in hypersensitivity reactions.^11,12

Through secretion of IL-4 and IL-6, activated basophils can cooperate with Th cells to induce immunoglobulin class switching in B lymphocytes, resulting in the secretion of various immunoglobulins, such as IgM, IgG, and IgA. Basophils can be activated by numerous cytokines including IL-17, IL-18, IL-26, and IL-33.^13,14 After basophils are activated, these cells constitutively express IL-4. Basophils are a potent source of IL-4, which represents the main cytokine promoting a Th2 lymphocyte polarization. ¹⁵ In the context of respiratory inflammation, some authors clearly showed in a murine experimental model that basophils are systemically induced upon allergenic stimulation and such an early basophil activation correlates with the Th2 lymphocytes, and basophils participate in the initiation of Th2 adaptive immune response. ¹⁶

Basophil activation mediated by pattern recognition receptors, Toll-like receptors (TLRs), has been extensively studied in recent years. Toll-like receptors are a family of transmembrane signaling receptors capable of selectively recognizing microbial components; this enables them to play an important role in innate immunity. ¹⁷ Toll-like receptors contain three domains: extracellular, transmembrane, and intracellular. After recognizing and binding to the corresponding pathogen-associated molecular patterns (PAMPs), TLRs activate intracellular signaling pathways that promote the release of various inflammatory factors and cytokines, thereby linking innate immunity to acquired immunity. The TLR family consists of 11 members; TLR2 is the most widely distributed TLR family member ¹⁸ and is primarily expressed on cells representing the first line of host defense, such as B lymphocytes, monocytes, macrophages, neutrophils, and basophils. TLR2 recognizes the most diverse molecules derived from pathogenic microorganisms and their products and functions in noninfectious inflammatory processes. Therefore, further studies on TLR2 have both theoretical and practical significance. Basophils can directly recognize various pathogenic microorganisms via TLRs and are involved in mediating the inflammatory responses.^19,20 For instance, peptidoglycans, which are TLR2 ligands, can induce the secretion of IL-4 and IL-13 by basophils in vitro. IL-4 can promote the maturation and differentiation of Th2 cells, thereby participating in the Th2 immune response. In addition, the IgE-mediated basophil activation also involves complement receptors (CRs), such as CR1, CR2, and CR3, which can induce histamine secretion by basophils.

CD63 was previously used as a marker of basophil activation. However, recent studies have shown that CD203 as a marker of basophil activation—provides greater sensitivity than CD63 in detecting basophil activation. ²¹ Currently, flow cytometry is often used to qualitatively determine the CD203c-positive cell count as well as to quantitatively measure the average fluorescence intensity of CD203c to evaluate the degree of basophil activation.

Promotion of Th2 responses

The cytokines secreted by activated basophils are involved in mediating the immune responses and immunomodulation. Notably, activated basophils can rapidly secrete IL-4 and IL-13.^22,23 IL-4 is considered the most important cytokine in the initiation and maintenance of Th2 cell differentiation. IL-4 can be secreted by various types of cells including Th2 cells, eosinophils, mast cells, and basophils that play a role in humoral immunity. IL-4 also plays an important role in regulating the stability and differentiation of Th2 cells and in the formation of memory Th2 cells. ²⁴ Additionally, Th2-type cytokines, such as IL-4 and interferon (IFN)-γ, are upregulated during the co-culture of antigen-stimulated CD4⁺ T cells and basophils in vitro; these cytokines facilitate the differentiation of CD4⁺ T cells into Th2 cells. ²⁵ IL-4 can also promote the differentiation of CD4⁺ T cells into Th2 cells, which secrete IL-4, IL-13, and other Th2-type cytokines. In the early stages of the immune response, re-exposure to antigens triggers the rapid secretion of large amounts of IL-4 by the basophils; in contrast, in late stages, IL-4 is primarily secreted by the memory T cells. Various exogenous lectins have the capacity to activate basophils. Moreover, various ligands, including bacterial peptidoglycans, viral proteins, and fungal mannans, can bind to TLR2 and promote the activation of basophils, which then produce IL-4 and promote the Th2 immune responses. Accordingly, basophils can aggravate the immune response triggered by viral and bacterial infections.^26,27 Hence, IL-4 secreted by the basophils in the early stage of the immune response plays a key role in initiating and maintaining the Th2 responses, which may accelerate disease progression.

Humoral responses

In humans, basophils serve as enhancers of the humoral response. Exogenous antigens bind to receptors expressed on the basophil surface via IgD and induce the production of IL-4 and B-cell activating factor (BAFF). Through these responses, activated basophils promote immunoglobulin class switching in B lymphocytes (which produce IgD and IgM in the early stages of differentiation), resulting in the secretion of large amounts of IgM, IgD, and IgA, thereby facilitating adaptive immune responses. ²⁸ The types of immunoglobulins elevated in the body may be associated with the types and quantities of the stimulating antigens. After initial exposure to an antigen, basophils express substantial amounts of specific IgE antibodies on their surfaces. After being re-exposed to the antigen, basophils can bind via CD40L to B cells expressing CD40 on their surface and induce the production of high concentrations of immunoglobulins by B cells. Moreover, activated basophils also produce large amounts of IL-4 and IL-6, which promote the differentiation of Th2 cells. Activated Th2 cells secrete IL-4, IL-5, IL-13, and IL-10, promote the proliferation and differentiation of B cells, and stimulate immunoglobulin production by B cells via binding to CD40 expressed on their surface through CD40L, thereby enhancing the humoral immune response. ²⁹

Immunomodulatory roles of basophils in SLE^{1–33,35–58}

Systemic lupus erythematosus is a complex disease that affects multiple organs and is associated with disorders of humoral immunity. Kidney injury occurs in more than 90% of SLE patients, primarily due to immune injury caused by the deposition of immune complexes containing IgM, IgG, and IgA in large quantities in the kidneys. These immune complexes comprise autoantibodies specific to nuclear components (antinuclear antibodies [ANAs]) or nucleic acids double-stranded DNA [dsDNA]). Many studies have evaluated the roles played by Th1, Th17, and regulatory T cells in the development of SLE.^30,31 Additionally, Th2 cells have been shown to have a role in the onset of SLE. ³² Several studies have also assessed the roles of Th2-type cytokines in the development of SLE, and IL-4 and IgE have been shown to play important roles in the pathogenesis of SLE. ³³

Autoreactive antibodies may mediate immune responses in the kidneys of patients with SLE, leading to renal failure and even death. Autoreactive IgE is responsible for the onset and progression of diseases, and this phenomenon involves basophil activation, induction of their homing to the lymph nodes, and promotion of Th2 cell differentiation and autoantibody production. Notably, activated basophils can mediate immune responses via IgE-dependent and IgE-independent pathways,^34,35 and basophils act as the main effector cells in allergic reactions by mediating hypersensitivity reactions via an IgE-dependent pathway. ³⁶ Previous studies have shown that IgE deficiency can delay the progression and alleviate the severity of diseases by decreasing the production of autoantibodies, thereby reducing organ damage. Studies performed in mouse models of SLE have revealed that IgE can significantly reduce the production of autoantibodies and the number of plasma B cells in mice; reductions in IgE significantly decrease the infiltration of immune cells, particularly monocytes, neutrophils, eosinophils, and basophils into organs, whereas IgE deficiency significantly reduces basophil activation in the spleens of mice with SLE.^37,38 IgE-positive patients with SLE exhibit significantly higher disease activity and higher expression of the basophil activation marker CD203c than IgE-negative patients with SLE. ³⁹ Thus, IgE autoreactivity is closely associated with basophil activation and may increase the level of disease activity. These findings indicate that basophils can promote the amplification of autoimmune and auto-inflammatory responses via IgE-dependent pathways.

Basophils and the Th2-type environment can promote the progression of SLE and aggravate the severity of lupus nephritis. Autoreactive IgE antibodies can activate basophils by binding to FcεRI receptors expressed on the surface of basophils in tyrosine-protein kinase-deficient aged mice with lupus-like disease. These mice exhibit continuous changes in Th2-type cytokine levels, significantly elevated IL-4 levels, high expression of CD62L (an adhesion molecule) ⁴⁰ and the MHC-II molecule human leukocyte antigen type DR (which is involved in self-antigen presentation) on basophils,^41,42 production of large amounts of anti-dsDNA antibodies and other ANAs, and have clearly visible immune-complex deposits in the glomeruli of the kidneys. In contrast, basophil-knockout mice show significantly reduced levels of ANAs and anti-dsDNA antibodies, lower proportions of splenic plasma cells, and alleviation of the pro-inflammatory environment in the kidneys. Although high-affinity FcsRI is expressed by basophils and mast cells, IL-4 and IgE-deficient mice exhibit significantly lower CD62L expression and reduced numbers of basophils in the lymph nodes and spleen. The detection of BAFF receptors expressed on the surface of basophils present in the lymph nodes has also indicated that lymph node basophils may affect the survival and differentiation of B cells. Basophils migrate from the peripheral blood to the lymph nodes and spleen via the expression of CD62L and MHC-II and enable the modulation of B-cell function by secreting cytokines and facilitating immune responses. Examination of clinical specimens has revealed that basophils and autoreactive IgE are important factors responsible for the occurrence of Th2-biased immune responses in patients with SLE. ⁴³ Basophils are activated (with high CD203c expression) in patients with SLE; hence, basophil activation may be an important feedback loop that affects the progression of SLE. Therefore, reducing the levels of IgE immune complexes in the blood or the degree of basophil activation may represent a new therapeutic approach for SLE.

Significant reductions in the levels of inflammatory factors (e.g., IL-1β, IL-4, IL-6, IL-13, and IFN-γ) in basophil-knockout mice with SLE have suggested that the alleviation of the pro-inflammatory environment in the kidneys via basophil knockout or reduction of basophil activity may be conducive to the treatment of SLE. ⁴⁴ Therefore, some currently available anti-allergic drugs (such as omalizumab) could exert therapeutic effects against SLE by reducing the IgE levels in the body and the expression of the FcεRI receptor on the surface of basophils. ⁴⁵

We have known the key role of IL-17 in tissue inflammation autoimmunity. It has been found that IL-17 produced by basophils plays an important role in the pathogenesis of lupus. Pan et al. found that the increase of serum autoantibodies in patients with lupus erythematosus is related to basophil activation. The serum levels of autoantibodies and IL-17 were decreased in basophil-depleted MRL-lpr/lpr mice. These findings suggest that basophil activation-dependent autoantibody and IL-17 production may constitute a critical pathogenic mechanism in SLE. ⁴⁶ Wakahara et al. found activated basophils amplified IL-17 release in effector memory T cells (TEM) and enhanced the ERK1/2 signaling pathway, which can result in chronic inflammatory disorders beyond Th2. ⁴⁷

The decreased number of basophils in the peripheral blood of patients with SLE may be attributed to multiple factors, such as inflammatory factor and chemokine-mediated recruitment of basophils from the peripheral blood into tissues. Basophil infiltration into these organs may result in damage to multiple systems, and SLE may therefore affect all body systems. Additionally, the observed apoptosis of T and B lymphocytes and production of pathogenic autoantibodies in large quantities during active SLE indicate that the number of basophils may also decrease due to excessive apoptosis.

Toll-like receptors 2 can mediate basophil activation. Pawar et al. found that mice with lupus nephritis have enlarged spleens and exhibit increased secretion of inflammatory factors (anti-dsDNA antibodies, IL-6, IL-12, and TNF-α), resulting in the exacerbation of the progression of lupus nephritis after exposure to bacterial cell wall components. ⁴⁸

Potential role of basophils in RA and other rheumatic diseases

Rheumatoid arthritis is a heterogeneous disease, that is, it cannot be attributed to a single cause. Several studies have evaluated the roles of various inflammatory factors, cytokines, and chemokines in the context of RA onset; however, the pathogenesis of this disease remains unclear. Different clinical subtypes of RA exhibit varying disease durations, prognostic outcomes, and therapeutic outcomes.

Many recent studies have assessed the role of Th1/Th2 imbalance in the onset of RA, ⁴⁹ with special focus on the detection of various cytokines and inflammatory factors in patients with RA. Notably, the dominant cytokines vary across different stages of the disease, resulting in differences in disease duration among patients with RA. In the early stages of the disease, the synovium and synovial fluid of patients with RA are dominated by Th2-type cytokines (IL-4 and IL-13), whereas the Th1-type cytokine IFN-γ dominates the synovium of patients with chronic RA in the late stages. ⁵⁰ Accordingly, many researchers have recently evaluated the physiological roles of basophils in regulating the Th1/Th2 balance. Using a basophil-deficient mouse model, researchers have demonstrated that IL-4 is mainly produced by basophils ⁵¹ and can induce the early differentiation of Th2 cells and cooperate with dendritic cells to regulate Th2-type immune responses by promoting the differentiation of Th2 cells; and thus is responsible for the development of Th2-associated diseases. ⁵² Basophils are activated in response to the specific binding of antigens with the IgE receptor FceRI expressed on its surface. Activated basophils can produce Th2-type cytokines, such as IL-4, which promote Th2 cell differentiation, enhance humoral immune responses, and affect the Th1/Th2 balance by reducing the Th1 response; this is the mechanism responsible for the pathogenesis of RA. Qiu Hua et al. ⁵³ have found high levels of IgE-containing circulating immune complexes (IgE-CICs) in the peripheral blood of patients with RA. Because they mainly enrolled older patients (49.5 ± 1.5 years) with longer disease durations, the disease had progressed to moderate and late stages in most patients. Importantly, they found that most patients with RA had elevated serum levels of IFN-γ, and there were no significant differences in the levels of IL-4 between groups with different disease activities, suggesting Th1 dominance in patients with chronic RA. Anti-cyclic citrullinated peptide (CCP) antibodies are relatively specific for RA and are produced by plasma cells in the synovium of patients with RA. Previous studies have shown that CCP can induce the activation of basophils by forming IgE-CICs with FceRI, which is highly expressed on the surface of basophils. Activated basophils release inflammatory factors, such as TNF-α and IL-1, thereby further aggravating RA severity, particularly in patients with severe arthritis and vasculitis complications. ⁵⁴

It has been found that basophils also play a role in other rheumatic diseases. TLR2 could mediate basophil activation that induces IgG4 production in IgG4-related disease. This enhancement of IgG4 production has been associated with BAFF and IL-13. ⁵⁵ Poddighe et al. found that the activation of basophils can induce autoimmune pancreatitis. ⁵⁶ In addition, recent studies have revealed autoimmune pancreatitis as the initial presentation of IgG4-related disease. Basophils release interferon-α and IL-33 after activation mediate autoimmune pancreatitis and human IgG4-related disease. ⁵⁷