Roles Of Collectins In Resisting Allergic Inflammation And Hypersensitivity Induced By Fungal Allergens And Antigens

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A. fumigatus causes systemic infection via the lungs (IPA or IA) in the immuno-suppressed subjects. However, in the immunocompetent individuals (probably with genetic susceptibility), it can cause an allergic disorder, called allergic bronchopulmonary aspergillosis (ABPA), which is different from other hypersen-sitivity responses to inhaled allergens in that the A. fumigatus spores grow in the respiratory tract and continually shed soluble and particulate antigens and allergens in the large subsegmental bronchi. Thus, the interaction of SP-A and SP-D with glycoprotein allergens of A. fumigatus, and the subsequent outcome of these interactions has been examined in vitro and in vivo (Madan et al. 1997b, 2001). SP-A and SP-D have been previously shown to bind allergens derived from pollen grains and dust mite (Kishore et al. 2002). SP-A, SP-D and rhSP-D can also bind to the three-week culture filtrate (3wcf) of A. fumigatus as well as purified glycoprotein allergens, gp55 and gp45, inhibit the ability of specific IgE to bind these allergens, and block histamine release from sensitised basophils isolated from ABPA patients (Madan et al. 1997b). Consistent with their roles in the modulation of allergic reactions, SP-A and SP-D have been reported to reduce the proliferation of PBMC isolated from dust mite-sensitive asthmatic children (Wang et al. 1998), and SP-D in particular has a suppressive effect on the secretions of IL-2 by PBMC (Borron et al. 1998). Furthermore, SP-A suppresses the production and release of IL-8 by ionomycin-stimulated eosinophils (Cheng et al. 1998). Since IgE cross-linking, histamine release and lymphocyte proliferation are essential immunologic steps in the development of A. fumigatus induced ABPA symptoms, SP-A and SP-D appear to be important in resisting allergenic challenge and dampening subsequent A. fumigatus induced hypersensitivity reactions in the lungs (Kishore et al. 2002,2005).

The therapeutic effects of intranasal administration of SP-A, SP-D or rhSP-D in a murine model of ABPA induced by A. fumigatus 3wcf using BALB/c strain of mice have been examined (Madan et al. 2001b). ABPA is an A. fumigatus-induced allergic disorder which is clinically characterised by episodic bronchial obstruction, positive immediate skin reactivity, elevated A. fumigatus-specific IgG and A. fumigatus-specific IgE antibodies in serum, peripheral and pulmonary eosinophilia, central bronchiectasis, and expectoration of brown plugs or flecks. The murine model resembled the human disease immunologically, exhibiting high levels of specific IgG and IgE, peripheral blood and pulmonary eosinophilia, and a Th2 cytokine response. Intranasal administration of SP-A, SP-D or rhSP-D (3 doses on consecutive days) significantly lowered eosinophilia and specific antibody levels. This therapeutic effect persisted up to 4 days in the SP-A treated ABPA mice, and up to 16 days in the SP-D or rhSP-D treated ABPA mice. Lung sections of the ABPA mice showed extensive infiltration of lymphocytes and eosinophils, which were considerably reduced following treatment. The levels of IL-2, IL-4 and IL-5 were decreased, while that of IFN-y was raised in supernatants of the cultured spleen cells, indicating a marked shift from pathogenic Th2 to a protective Th1 polarisation of helper T cell (Th) immune response.

In view of the proposed role of SP-D in regulation of A. fumigatus mediated allergic hypersensitivity, Atochina et al. (2003) hypothesised that an elevated SP-D production is associated with the impaired ability of C57BL/6 mice to develop airway hyper-responsiveness (AHR) to A. fumigatus challenge. The allergen challenge to sensitised C57BL/6 mice induced a markedly increased SP-D protein expression in the surfactant fraction (1, 894 ± 170% of naïve controls) that was 1.5 fold greater than the increase in Balb/c mice (1,234± 121%). In addition, sensitised and exposed C57BL/6 mice had significantly lower IL-4 and IL-5 in the BALF than Balb/c mice (p < 0.05), suggesting that enhanced SP-D production in the lung of C57BL/6 mice may contribute to an attenuated AHR in response to allergic airway sensitisation. Thus, SP-D may act by inhibiting production of Th2 cytokines.

It is evident that SP-A and SP-D appear to offer protection against aller-genic challenge at various levels, suggesting a hierarchical role for these two molecules of innate immunity. These protective mechanisms seem to involve allergen scavenging that leads to inhibition of allergen-IgE cross-linking and histamine release, suppression of the activation of sensitised basophils, mast cells or eosinophils, suppression of B and T cell proliferation, modulation of DCs and macrophages, and Th cell polarisation (reviewed in Kishore et al. 2002; Sonar et al. 2006). The ability of SP-A and SP-D to suppress proliferation of specific B-lymphocytes may account for the lowering of specific IgG and IgE levels in the treated group of allergic mice; this effect may well be amplified by a decrease in IL-2 levels since IL-2 is central to lymphocyte growth and differentiation. IL-5 is a differentiation factor for eosinophils whereas IL-4, together with IL-13, is an important factor for isotype switching of B-lymphocytes, leading to the secretion of IgG1 and IgE. These cytokine profiles mark a characteristic Th2 response in the allergic immune reaction that is characterised by secretion of IL-4, IL-5, IL-10 and IL-13 and generation of humoral immune responses. Shifting of cellular responses from a predominantly Th2 to a Th1 cytokine profile, following treatment with SP-A, SP-D or rhSP-D, appears central to the protective mechanism since IFN-7, a Th1 cytokine, promotes cellular immunity and normally inhibits Th2 differentiation in response to IL-4. Among the factors that have been shown to influence the Th1-Th2 balance, IL-12 is dominant in directing the development of Th1 cells that produce high amounts of IFN-7. Thus, SP-A and SP-D have been shown to modulate DCs differentially. The SP-D mediated binding and uptake of E. coli by bone-marrow derived mouse DCs has been shown to increase antigen presentation of E. coli expressed proteins to T-cell hybridoma (Brinker et al. 2001)). Curiously, pre-treatment of immature DCs with SP-A (or C1q) has been shown to inhibit LPS-mediated surface expression of maturation markers: MHC class II and CD86. Stimulation of immature DCs by SP-A also inhibits the allostimulation of T cells and enhances dextran endocytosis (Brinker et al. 2003). These results appear to suggest an immune balancing role for SP-A and SP-D during pulmonary inflammation (Kishore et al. 2006).

Since complement activation contributes to the pathogenesis of allergy, the role of MBL in ABPA pathogenesis has recently been assessed (Kaur et al. 2006). Significantly higher MBL levels and activity were observed in the allergic patients as compared to the controls (44 patients of bronchial asthma with allergic rhinitis, 11 ABPA patients, and 40 unrelated, age-matched controls of Indian origin). High MBL activity showed a positive correlation with peripheral blood eosinophil counts in the allergic patients (r = 0.75). The levels of the two mouse MBLs (MBL-A and MBL-C) were evaluated in mice before and after sensitisation with allergens and antigens of A. fumigatus. The MBL-A levels were significantly higher in the mice after their sensitisation with fungal allergens (p < 0.05). In view of genetic polymorphisms in the collagen region of MBL gene contributing to varied plasma MBL levels and activity, single nucleotide polymorphisms (SNPs) in exon 1 and intron 1 (encoding the collagen region) of MBL in these two patient groups were examined. One of the intronic SNP G1011A showed significant association with both categories of allergic patients in comparison to the controls. The intronic SNP also showed a significant association with elevated peripheral blood eosinophil counts and a decreased percent-predicted FEV1 of the patients, the two commonly used markers of allergic airway diseases (Kaur et al. 2006). It appears that allergic patients with '1011A' allele and high plasma MBL levels and complement activity may be susceptible to a severe form of respiratory allergic disease.

8. SUSCEPTIBILITY OF SP-A" " OR SP-D" " MICE TO FUNGAL ANTIGENS AND ALLERGENS

The susceptibility of SP-A-/- or SP-D-/- mice to the A. fumigatus allergen challenge, as compared to the wild-type mice has been examined recently (Madan et al. 2005). Both SP-A-/- or SP-D-/- mice show intrinsic hyper-eosinophilia and several fold increase in lung levels of IL-5 and IL-13, with a lowering of the IFN-7 to IL-4 ratio in the lungs, suggesting an inherent bias to a Th2 immune response in the gene-deficient phenotype as compared to the wild type mice. Treating SP-A-/-, or SP-D-/-, mice with SP-A or SP-D, respectively, reduces this hyper-eosinophilia and Th2 predominance. The SP-A-/-, and SP-D-/- mice show distinct immune responses to A. fumigatus sensitisation, SP-D-/- mice being more susceptible than wildtype mice to pulmonary hypersensitivity induced by being A. fumigatus allergens. Interestingly, SP-A-/- mice have been found to be nearly resistant to A. fumigatus sensitisation. Intranasal treatment with SP-D or rhSP-D can rescue the A. fumigatus sensitised SP-D-/- mice, while SP-A treated A. fumigatus sensitised SP-A-/- mice show several fold elevated levels of IL-13 and IL-5, resulting in increased pulmonary eosinophilia and damaged lung tissue. This validates important roles for SP-A and SP-D in the modulation of pulmonary hypersensitivity and suggests differential mechanisms involved in SP-A and SP-D mediated resistance to allergen challenge. Hyper-eosinophilia exhibited by both SP-A-/- and SP-D-/- mice, probably due to significantly raised levels of IL-5 and IL-13 in these mice, suggests that SP-A and SP-D have a role in regulating eosinophil infiltration and modulation in the lung in response to environmental stimuli. It is interesting to note that similar to SP-D-/-

mice, IL-13 over-expressing mice have characteristic foamy macrophages, type II cell hypertrophy, fibrosis, massive inflammation involving eosinophilia, protease-dependent acquired emphysema, and airway hyperresponsiveness (AHR) (Homer et al, 2002). Given the involvement of IL-13 in processes such as mucus production and AHR, as well as eosinophil survival, activation and recruitment, it is likely that certain physiological effects in SP-A-/- as well as SP-D-/- mice arise due to over-expression of IL-13 (Homer et al. 2002, Madan et al. 2005). It is also evident that SP-A and SP-D inhibit allergen-mediated eosinophilia in the lungs through down-regulation of IL-5. SP-A and SP-D have important roles in the regulation of the cytokine milieu and eosinophilia in the lungs, and the inherent hypersensitivity due to their deficiency in the SP-A-/- and SP-D-/- mice argues for it.

9. PENTRAXINS AS ADAPTORS BETWEEN INNATE AND ADAPTIVE IMMUNITY

Pentraxins are characterised by the presence, in their carboxy-terminus, of a 200 amino acid pentraxin domain, with an 8 amino acid long conserved pentraxin signature sequence (HxCxS/TWxS, where x is any amino acid) (Figure 2). CRP and SAP are classic short pentraxins produced in the liver. CRP is produced as a nonspecific acute phase reactant to inflammation, infection and tissue injury. CRP levels in the plasma of healthy adults are barely detectable but can potentially increase as much as 10,000-fold following an acute phase stimulus as a result of accelerated rates of transcription in the liver (Pepys and Hirschfield 2003). Circulating CRP is produced only by hepatocytes, mainly in response to the proinflammatory cytokine IL-6, but lymphocytes and monocytes/macrophages are also able to synthesise CRP. SAP, a basement membrane component, is the main acute phase protein in mice, whereas in human serum it is constitutively present at 30-50 ^g/ml. Long pentraxins are expressed in a variety of tissues including CNS. PTX3, a prototypical long pentraxin, is produced in response to pro-inflammatory cytokines, most abundantly by DCs. As shown in Table 2, CRP, SAP and PTX3 have diverse and important functions in immunity. Recently, PTX3 has been shown to have a nonredundant role in resistance against A. fumigatus.

10. ANTI-MICROBIAL FUNCTIONS OF SHORT PENTRAXINS: CRP AND SAP

The physiological functions of CRP and SAP involve calcium dependent binding to a range of ligands (Table 2). CRP binds to phosphorylcholine (PC), a major constituent of C-type capsule polysaccharides of Streptococcus pneumoniae, as well as various pathogens including bacteria, yeasts and fungi (Szalai 2002). CRP has been shown to act as an opsonin for the phagocytosis of attenuated strains of C. albicans blastopores and thus may offer protection against candi-dosis independent of complement (Richardson et al. 1991a). However, virulent strains appear to be less sensitive to CRP-mediated phagocytosis and subsequent

Figure 2. (a) Organisation of short and long penraxins. Pentraxins are characterised by the presence in their carboxy-terminal of a 200 amino acids pentraxin domain, with an 8 amino acid long conserved pentraxin signature (HxCxS/TWxS, where x is any amino acid). The human CRP and SAP genes are located on chromosome 1q23 and are organised in two exons, the second exon encoding for the pentraxin domain. The long pentraxin, human PTX3 gene, localised on human chromosome 3 band q25, is organised in three exons separated by two introns, the third exon codes for the pentraxin domain. The mature SAP protomer is 204 amino acid long (25,462 Da) and has a pentameric structure in the presence of physiological levels of calcium (127,310 Da). In the absence of calcium, SAP consists of both pentameric and decameric forms. Each SAP protomer is glycosylated with a single N-linked biantennary oligosaccharide at Asn32. Human CRP is composed of five identical nonglycosylated protomers. The PTX3 protein (40,165 Da) consists of a C-terminal 203 amino acids pentraxin-like domain (containing an N-linked glycosylation site in the C-terminal domain at Asn220) and an additional N-terminal region (178 aa) unrelated to other known proteins. PTX3 protomers can assemble as decamers and higher oligomers upto 900 kDa. (b) Crystal structures of CRP and SAP. Each CRP protomer has a characteristic lectin fold composed of two layered B sheets with a flattened jellyroll topology; five protomers are noncovalently

Figure 2. (a) Organisation of short and long penraxins. Pentraxins are characterised by the presence in their carboxy-terminal of a 200 amino acids pentraxin domain, with an 8 amino acid long conserved pentraxin signature (HxCxS/TWxS, where x is any amino acid). The human CRP and SAP genes are located on chromosome 1q23 and are organised in two exons, the second exon encoding for the pentraxin domain. The long pentraxin, human PTX3 gene, localised on human chromosome 3 band q25, is organised in three exons separated by two introns, the third exon codes for the pentraxin domain. The mature SAP protomer is 204 amino acid long (25,462 Da) and has a pentameric structure in the presence of physiological levels of calcium (127,310 Da). In the absence of calcium, SAP consists of both pentameric and decameric forms. Each SAP protomer is glycosylated with a single N-linked biantennary oligosaccharide at Asn32. Human CRP is composed of five identical nonglycosylated protomers. The PTX3 protein (40,165 Da) consists of a C-terminal 203 amino acids pentraxin-like domain (containing an N-linked glycosylation site in the C-terminal domain at Asn220) and an additional N-terminal region (178 aa) unrelated to other known proteins. PTX3 protomers can assemble as decamers and higher oligomers upto 900 kDa. (b) Crystal structures of CRP and SAP. Each CRP protomer has a characteristic lectin fold composed of two layered B sheets with a flattened jellyroll topology; five protomers are noncovalently killing by human neutrophils (only 10% killing). CRP has also been shown to bind A. fumigatus conidia and enhance phagocytosis by neutrophils without requiring participation of complement components (Richardson et al. 1991b). In an earlier study (Jensen et al. 1986), CRP was found to bind hydrophobic fractions containing phosphorylcholine from A. fumigatus hyphal homogenate in a calcium-dependent manner, suggesting that CRP may modulate immune response to antigen and allergens of A. fumigatus.

Despite extensive biochemical, structural and diagnostic characterisation of CRP and SAP, their in vivo functions are still debated. Transgenic and gene-deficient mice have also been used to identify biological functions. However, the two proteins are regulated differently in human and mouse. Administration of human CRP to the transgenic mice increases survival extent and time of mice infected with Streptococcus pneumoniae (Szalai et al. 1995). Similar protective effects of CRP overexpression have been noted against Haemophilus influenzae and Salmonella enterica (Weiser et al. 1998, Szalai et al. 2000). Studies using SAP-/- mice have revealed that SAP plays a dual role in bacterial infections (Noursadeghi et al. 2000). SAP binds to Streptococcus pyogenes, Neisseria meningitidis and the rough variant of E. coli, and shows anti-opsonic effect, thus reducing phagocytosis and killing by neutrophils. SAP-/- mice survive an otherwise fatal challenge with S. pyogenes and rough E. coli J5. However, SAP-/- mice are more susceptible to a non-binding smooth strain of E. coli. Thus, SAP offers protection against non-binder pathogens such as the smooth variant E. coli, while a strong anti-opsonic effect is observed when SAP binds to bacteria, resulting in enhanced virulence of the infectious agent. Such interesting studies have not been carried out using fungal pathogens.

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Coping with Asthma

Coping with Asthma

If you suffer with asthma, you will no doubt be familiar with the uncomfortable sensations as your bronchial tubes begin to narrow and your muscles around them start to tighten. A sticky mucus known as phlegm begins to produce and increase within your bronchial tubes and you begin to wheeze, cough and struggle to breathe.

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