Complement components as potential therapeutic targets for asthma treatment

Summary

Asthma is the most common respiratory disorder, and is characterized by distal airway inflammation and hyperresponsiveness. This disease challenges human health because of its increasing prevalence, severity, morbidity, and the lack of a proper and complete cure. Asthma is characterized by T_H2–skewed inflammation with elevated pulmonary levels of IL-4, IL-5, and IL-13 levels. Although there are early forays into targeting T_H2 immunity, less-specific corticosteroid therapy remains the immunomodulator of choice. Innate immune injury mediated by complement components also act as potent mediators of the allergic inflammatory responses and offer a new and exciting possibility for asthma immunotherapy. The complement cascade consists of a number of plasma- and membrane-bound proteins, and the cleavage products of these proteins (C3 and C5) regulate the magnitude of adaptive immune responses. Complement protein are responsible for many pathophysiological features of asthma, including inflammatory cell infiltration, mucus secretion, increases in vascular permeability, and smooth muscle cell contraction. This review highlights the complement-mediated injury during asthma inflammation, and how blockade of active complement mediators may have therapeutic application.

Introduction

Asthma is a chronic inflammatory disease of the bronchi arising because of inappropriate immunological responses to common environmental antigens in genetically susceptible individuals [1]. It is thought to be mediated by CD4⁺ T lymphocytes that produce T_H2 cytokines linked with elevated specific IgE, eosinophilia, and airway hyperresponsiveness (AHR) [2–4]. This perspective will explore how an important component of the innate immunity, the complement system, normally a key defense against mucosal bacteria, viruses, fungi, helminthes, and other pathogens, may also play an important role in the pathogenesis of asthma. Although complement factors have been associated with development of pathophysiology of asthma [5, 6], the role of individual complement components in the pathogenesis of allergic asthma is not clear. Biologically active fragments (C3a, C5a), generated through the classical, alternative, lectin pathways, and by the direct action of certain proteolytic enzymes on C3 or C5 [7] (Fig. 1), participate in AHR induction. Infections, and allergens of respiratory tract activate local complement activation participate in AHR [8–10] because of their ability to recruit, activate leukocytes, increase vascular permeability, stimulate contraction of smooth muscle, and trigger degranulation of mast cells [9, 11–13]. In addition to allergens, other triggers of asthma have been shown to activate complement cascade in human, and in animal models [13]. It has been demonstrated that bronchoalveolar lavage (BAL) of asthma individuals contain quantitatively higher levels of C3a and C5a as compared to healthy control subjects at baseline [14].

Figure 1.

Model explains the generation of C3a and C5a through classical, lectin and alternative pathway during airway inflammation. Further, C3a binds to C4aR on CD4⁺ T cells and promotes recruitment of IL-17⁺CD4⁺ cells, neutrophil inflammation and activation of Mast cells that leads to histamine mediated AHR.

In asthma, overproduction of activated complement fragments may promote asthma susceptibility [13]. This imbalance results in up regulation of biologically active fragments, C3a and C5a, which may act on cells of the innate immune system to favor asthma development [9, 11]. The anaphylatoxins C3a and C5a have been characterized as potent mediators of the effector phase of the allergic response [8–10, 15] with C3a regulating T_H2 cytokine production possibly through the recruitment, and activation of T_H2 cells [13]. C5a plays a dual immunoregulatory role by protecting against the T_H2-polarized adaptive immune response and mediates type 2 inflammatory responses once inflammation proceeds [13] (see Fig. 2). Complement may participate in the development of susceptibility to asthma, despite a normal level of complement fragments generated during complement activation.

Figure 2.

Model explains Th1 to Th2 shift during the development of asthma pathogenesis, and, C3a and C5a as a potential targets to rescue asthma by blocking local T cell recruitment.

Different models of experimental allergic asthma suggest that the C3a and C5a not only promote pro-allergic effector functions during the allergic effector phase, but also regulate the development of T_H2 immunity during allergen sensitization [16]. Generation of C3a on airway surfaces induce T_H2-mediated inflammatory responses to a variety of environmental triggers of asthma (i.e., allergens, pollutants, viral infections, cigarette smoke) [9, 11]. C5a is dominant during allergen sensitization, and protects against the development of maladaptive T_H2 immunity [13, 16]. By contrast, C3a and C5a appear to act synergistically and drive allergic inflammation during the effector phase [10]. In addition to its proinflammatory effector functions, complement regulates adaptive immunity at many levels [12], and play critical role as well in causing vascular injury in allografts [17, 18].

It has been observed that allergen challenged C3aR-deficient mice and guinea pigs are protected against bronchoconstriction and AHR [16]. Interestingly, there was no difference in eosinophilic airway inflammation, T_H2 cytokine production, IgE production between C3a receptor-deficient, and in wild type animals which demonstrate that airway inflammation, and AHR are two independent features of asthma [2, 3]. Several studies have demonstrated that blocking of IL-4 reduces AHR in the lung, and that RAG^−/− mice, which lack Th2 cells, fail to develop AHR, mucus hyper-secretion, and eosinophilia during the course of asthma [19]. However, airway inflammation, and the immune responses at cellular and molecular levels have led to the proposition of a number of mechanisms such as mast cell degranulation [18, 20, 21], neurogenic dysfunction, involvement of T-lymphocytes, eosinophils, altered immunosuppressive macrophages, excessive nitric oxide through inducible nitric oxide synthase, overproduction of proinflammatory cytokines and immunoglobulins [22] during the asthma development.

Asthmatic inflammation may be initiated or exacerbated by amplification of the complement cascade [11–13]. Complement components, especially C5 and C3 with their associated cleavage products C5a and C3a, regulate the magnitude of adaptive immune responses via ligation of their respective receptors expressed on antigen-presenting cells, and T lymphocytes, as well as on pulmonary structures, and stromal cells [5, 22]. These immune responses involve many pathophysiological features of asthma that include inflammatory cell infiltration, mucus secretion, increase vascular permeability, and smooth muscle contraction [23]. This review summarizes the crucial role of complement mediators in airway inflammation, and how it affects the pathogenesis of asthma disease.

Generation of c3a and c5a in asthma

Asthma is associated with activation of complement cascade and allergen induced complement generates C3a and C5a [3]. It has been demonstrated that C3a plays a crucial role in asthma primarily by regulating mast cell-ASM (Airway Smooth Muscle) cell interaction [14]. C3a and C5a are released as key active factors in complement cascade that modulate innate immunity [3, 4]. C5a is, however, involved in a number of inflammatory diseases [24] such as immune-complex-mediated lung injury, microvascular injury in rejecting allografts [20] and in sepsis [14]. Levels of C3a are found elevated in bronchoalveolar lavage fluid after allergen challenge in asthmatic but not among healthy controls [3]. The C3a and C5a peptides regulate inflammatory functions by interacting with their receptors C3aR and C5aR [25, 26]. These receptors were mostly present only on myeloid cells such as macrophages, neutrophils, eosinophils, basophils, and mast cells, however, the immune cells that express these receptors in the lung have been investigated, and their expression been examined during phase of asthma inflammation [27–30]. These findings suggests the participation of bronchial epithelial and smooth muscle cells in the pathology of diseases such as sepsis and asthma, the data suggest a role for complement receptors during lung inflammation [27].

It has been observed that C3aR activation is associated with the development of AHR, and inflammation in different animal models of asthma [27]. However, C3aRdeficient mice are protected from AHR in response to aerosolized ovalbumin challenge following intraperitoneal sensitization with ovalbumin [31]. Single nucleotide polymorphisms in C3 and C3aR genes have been linked with increased susceptibility to asthma [31]. This speculates the crucial role of C3a and C3aR in the development of AHR and inflammation [31]. BAL fluids of C3aR deficient mice also had low levels of T_H2 cytokines (IL-4, IL-5, and IL-13), IgE titers, and mucous production that further support a role of C3a receptors in the development of AHR, and generalized inflammation [15, 31, 32]. It is observed that deficiency of C3aR leads to decrease airway hyperresponsiveness in a mouse model pulmonary allergy [32]. In addition, increased C3a levels have been reported in bronchial lavage samples from allergen-challenged asthma patients [3]. There is a significant association has been reported between AHR and C5 level [3], however, compared to C5 sufficient mice, the C5-deficient mice are more responsive to methacholine challenges after allergen exposure [33]. The presence of C5 and C5aR is necessary for a variety of immunological responses including inflammation and host defense [14]. Elevated levels of complement anaphylatoxin peptides have been observed in the lungs of asthmatic patients [27] which further supports the significance of complement factors in asthma pathogenesis. The C5 gene and the C5aR receptor genetic regions have been identified as putative asthma susceptible loci [27]. Finally, C3aR and C5aR expression has demonstrated on lung bronchial smooth muscle cells implicating these receptors as mediators of bronchoconstriction [34].

Complement mediators-immune cell interaction in asthma pathogenesis

Cells of the innate immune system in asthmatics are abnormally responsive to the regulatory effects of complement followed by the development of susceptibility to asthma [10, 35]. Recent research efforts have also demonstrated that CD4⁺ T cells, which produce a T_H2 pattern of cytokines, play a pivotal role in the pathogenesis of this disease [52] and cytokines such as IL-4, IL-13, and IL-5 to contribute in bronchial hyper-reactivity, and mucus hyper-secretion as well as orchestrate the recruitment, activation of mast cells, and eosinophils [19, 53, 54]. The complement cascade is a central player of innate immunity that coordinates a number of inflammatory responses [35]. C3a activates mast cells, basophils, eosinophils, and contraction of airway smooth muscle cell [18, 24]. Both C3a and C5a can induce ASM cell contraction, increase the microvascular permeability, and regulate vasodilation [5]. C5a has been widely used as standard stimulant to eosinophil/or basophil responsivity, and active C5 fragments alone can induce airway hyperresponsiveness when administered [24]. In addition, C3a and C5a can: 1) stimulate respiratory burst in macrophages, neutrophils, and eosinophils; 2) stimulate the release of histamine from basophils and mast cells; and 3) regulate the synthesis of eosinophil cationic proteins and adhesion to endothelial cells by eosinophils [14, 20]. C3a can also stimulate serotonin release from platelets, and modulate synthesis of IL-6 and TNF-α by B-lymphocytes and monocytes [36, 37]. C5a is a potent chemotactic molecule for macrophages, neutrophils, T lymphocytes, and basophils [27]. Both C3a and C5a can induce chemotaxis of eosinophils and mast cells [27]. Generation of C3a at the airway surface triggers induction of AHR [13], while C5/ C5a, plays a dual immunoregulatory role by protecting against the initiation of Th2-mediated immune responses during initial allergen exposure by its ability to affect dendritic cell-T cell interactions, and a more traditional pro-inflammatory role once immune responses are established [13, 16].

The interactions between C5a and the IL-12 are important for generating AHR-associated inflammation [38]. Direct administration of IL-12 has shown to reduce AHR, and macrophages from the C5-deficient mice were here shown to produce lower levels than the control mice [39–42]. C3b on the other hand, has been shown as capable of blocking IL-12 production by its interaction with the α_Mβ₂ integrin, an action perfectly in keeping with a positive role for C3 cleavage in AHR e either via C3a and its receptor or through C3b and reduced levels of IL-12 [43].

C5 has been associated with dendritic cells mediated induction of Tregs (CD4⁺CD25⁺ T cells) and Tregs blockade in allergen exposed C5 sufficient mice eliminated their protection from the development of AHR associated with a drop in the numbers of pulmonary dendritic cells [13]. In addition, depletion of dendritic cells and Tregs in mice results in an increased capacity to stimulate T cell proliferation and Th2 cytokine production. The balance between C3a and C5a during early life exposures to allergens may be a crucial determinant factor in the development of tolerance to inhaled antigens [9, 13]. In lungs, C3 would most probably create Th2 shift, which is consistent with data suggesting that the lungs have Th2 type cell at birth in newborn [44]. Clinical studies has shown the relatively higher levels of C3a and C5a in BAL fluid of allergen induced asthmatic airways as compared with control subjects [5, 10, 16]. C5a contribute to the development of the proallergic environment in allergic asthma [10], and targeting C5 in allergen-induced asthma model have demonstrated that C5 may serve as a suitable target in treatment of asthma [10, 45].

C5a can bind to both C5aR and C5L2 receptors [5], and, more specifically C5L2 acts at the dendritic cell and T cells interface, and control the development of T_H1 and T_H17 cells in response to airway antigen exposure, and drives T_H2 immune responses independent of specific dendritic cells [46]. As reported earlier, C5a, and perhaps C3a may cause immediate airflow obstruction, and subsequent airway hyperactivity [27]. It has been demonstrated in murine model of AHR that C5a may act directly or indirectly to stimulate C5aR on local mast cells and/or platelets, resulting in the release of broncho constrictive mediators, and results in sensitization of the airways without cellular inflammation [47]. In a number of other asthma models, the role of IL-17 has been highlighted in inducing asthmatic response, and AHR [48]. There has been increasing evidence suggest the involvement of C3a in the asthma pathogenesis, and the relationship between C3a driven IL-17 and IgE-mediated asthmatic responses that have shown the contribution of IL-17 to an IgE-mediated late-phase asthmatic response, and AHR [48]. They reported that during repeated antigen exposure, C3a mediated antibody production (IgE) results in production of IL-17⁺CD4⁺ cells in the lungs [18, 24, 49] (Fig. 1).

Summary

Asthma, a complex airway inflammatory disease, is characterized by bronchoconstriction, AHR and airway remodelling [50]. Current consensus suggests that T_H2 cytokine producing T cells, mast cells, and ASM cells play central roles in the pathogenesis of asthma [51]. This classification of asthma has led to the concept that the immediate response after allergen challenge is mediated by mast cells, whereas eosinophils are the predominant effector cells in the late asthmatic reaction [27]. C3 and C5 play unique roles in airway inflammation associated with asthma and the release of C3a at the airway surface mediates the induction of AHR in different asthma models, while C5/C5a plays a dual immunoregulatory role by protecting against Th2-mediated immune responses during initiation of responses, and a proinflammatory role once immune responses are established [50]. Serine proteases generated in response to classical and alternative pathways has potential to generate C3a and C5a from C3 and C5 respectively [55, 56]. It is observed that different components of the complement cascade have implicated in mediating allergic inflammation [57]. As reported in other asthma models, C3a and C5a participate in shifting Th2 and Th1 balance respectively but blocking or antagonizing C5a shift response to Th2 [13] and Th2 shift results in elevated Th2 adaptive response followed by airway inflammation [42]. These anaphylatoxins can induce ASM contraction [20, 58, 59], mucus secretion [60, 61], increased microvascular permeability [62, 63] [17, 24], vasodilation [64, 65], leukocyte migration and activation, and degranulation of mast cells [66], which are the hallmarks features of asthma. Further, the most important, neutralization of anaphylatoxin activity through the use of blocking antibodies, genetic targeting or by using specific antagonist of various complement factors or their receptors has been shown to attenuate allergic inflammation, and AHR in mice, and guinea pigs [67, 68]. It is becoming increasingly clear that immunoregulatory events occurring at the interface of innate and adaptive immunity play an important role in asthma pathogenesis [13]. The data reviewed here suggest that the complement pathway serves as a central regulator of adaptive immune responses to a variety of inhaled substances.

The complement cascade consists of number of serum and cellular proteins, and the activation of complement includes a series of initiation, amplification, and release of active mediators that mediate cell lysis [35]. The whole complement cascade is regulated at various points by different complement regulatory proteins. These proteins counter check the over expression of released active fragments, make balance between self and foreign tissue, and, therefore, allows for control over the potent tissue-damaging capabilities of complement activation. Soluble, and membrane-bound complement regulators have been produced, and shown to be effective in blocking complement activation in vitro as well as in animal models of complement-mediated pathologies in different diseases [69]. Compared with conventional therapeutic options available to asthma patients, recombinant proteins for therapy remain attractive to date, for reasons having to do with both the biological properties of proteins, and the economics of drug development [18, 24]. A number of complement inhibitors has been introduced as therapeutic agents for inflammatory, ischemic [70], and autoimmune diseases [42]. It has been reported that Crry-Ig treatment inhibit airway inflammation, and AHR in OVA sensitized mice [9, 11]. The objective here is to present a brief and selective summary of the findings using synthetic molecules for the therapeutic inhibition of complement in asthma pathogenesis. In last couple of years, it has been recognized that some of the endogenous complement regulatory proteins has been proven to serve as potential therapeutic agents in blocking inappropriate activation of complement in human diseases specially asthma [34, 45, 71, 72]. In this review, our aim is to focus on more translational approach in the field of asthma cure with the possible use of novel complement inhibition approach to control complement mediated airway injury, hyperresponsiveness and ultimately to rescue asthma.

Abbreviations

AHR

airway hyperresponsiveness

BAL

bronchoalveolar lavage

ASM

airway smooth muscle

MAC

membrane attack complex

Treg

regulatory T cells