The data presented in the current report demonstrate that anti-SP-R210 antibodies, α-SP-R210n and α-SP-R210ct, bind to more than 90% of CD14+ monocytes and less than 10% of CD3+ cells in freshly isolated PBMC. Stimulation of PBMC from PPD+ donors with heat-killed M. tuberculosis markedly increased the binding of antibodies to CD3+ cells, and this was correlated with increased expression of SP-R210 in PBMC after stimulation. The antibody, α-SP-R210n, inhibited proliferation of lymphocytes and secretion of IFN-γ by PBMC from healthy PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and the antibody α-SP-R210ct had no effect. Inhibition of IFN-γ production depended on the presence of APC. The antibodies against SP-R210n also inhibited M. tuberculosis-induced production of TNF-αand enhanced production of IL-10, and inhibition of IFN-γ production by α-SP-R210n was abrogated by neutralization of IL-10 and TGF-β1 simultaneously. As our studies were performed with polyclonal rather than mAb to SP-R210, we cannot exclude with certainty the possibility that some of the effects observed may be a result of binding of these polyclonal antibodies to unintended targets. Future studies with monoclonal reagents would be helpful to resolve this issue. Nevertheless, our current findings indicate that SP-A, via SP-R210, suppresses cell-mediated, immune responses to M. tuberculosis by enhanced production of IL-10 and TGF-β1 by APC.
Studies in animals and humans, using in vitro and in vivo models, have demonstrated that SPs, especially SP-A, exhibit potent regulatory effects on immunity and inflammatory reactions in the lung [1
]. Although the effect of SP-A on innate immunity has been studied extensively [1
], its effect on adaptive immunity in humans has been largely limited to studies of cell proliferation and IL-2 secretion in response to mitogens and anti-CD3 mAb [18
]. In the current report, we evaluated the effects of SP-A in a more physiologically relevant recall immune response to microbial antigen by human primary T cells from healthy persons infected with M. tuberculosis
. We found that the antibodies targeted against the SP-A-binding domain (α-SP-R210n) markedly inhibited M. tuberculosis
-induced proliferation of lymphocytes and secretion of IFN-γ and TNF-α in a dose-dependent manner ( and ). Anti-SP-R210n also elicited production of high concentrations of the anti-inflammatory cytokine IL-10, and the combination of neutralizing antibodies to IL-10 and TGF-β1 abrogated the inhibitory effects of α-SP-R210n (). Previous studies showed that SP-A induced TGF-β1 production by alveolar macrophages [11
], whereas the effect of SP-A on IL-10 production is more controversial. Chignard and colleagues [38
] showed that SP-A inhibited IL-10 production by LPS-stimulated murine alveolar macrophages, and the same group also reported that SP-A stimulated IL-10 production by bone marrow macrophages and U937 cells [40
]. In another study, SP-A-mediated uptake of respiratory syncytial virus suppressed virus-induced IL-10 secretion by primary monocytes and U937 monocytic cells [41
]. Importantly, SP-A-deficient mice had reduced the Th2 response and IL-10 secretion following pulmonary infection with influenza A virus, compared with wild-type animals [14
], indicating that SP-A regulates secretion of IL-10 in vivo. Underlying these results is the ability of SP-A to interact with pathogens and host cells, acting to block or stimulate pathogen-induced responses on one hand and modulating the inflammatory response of immune cells on the other [1
]. Here, direct probing of the functional domain of the SP-A receptor SP-R210 with an antibody revealed that this receptor inhibits Th1 immune responses. The current findings suggest that SP-A binds to SP-R210n, which is present on the vast majority of monocytes, eliciting production of IL-10 and TGF-β1, known to inhibit production of IFN-γ by reducing the capacity of APC to produce IL-12 and to express costimulatory molecules [42
]. Our results extend the findings of previous reports that SP-A inhibits expression of co-stimulatory molecules on murine bone marrow-derived dendritic cells and reduces their capacity to support T cell proliferation in response to antigen [16
We found that the inhibitory effects of α-SP-R210n on production of M. tuberculosis
-induced IFN-γ depended on the presence of monocytes. Although SP-A is primarily produced by airway epithelial cells, many reports document that SP-A is present in multiple tissues, including the gastrointestinal and sinus mucosa, peritoneum, pericardium, and skin [44
]. SP-A is also present in plasma at low levels, and these levels increase in patients with lung diseases such as pulmonary fibrosis and acute lung injury [48
]. We speculate that SP-A plasma levels may be elevated in chronic lung infections such as tuberculosis and that this may contribute to the reduced production of IFN-γ by peripheral blood T cells in response to mycobacterial antigens in tuberculosis patients [33
The lung is continually exposed to foreign antigens, and uncontrolled inflammatory responses to these antigens could cause significant tissue damage and reduced gas exchange. Alveolar macrophages have potent phagocytic activity but low immunostimulatory capacity and inhibit the response of alveolar T lymphocytes to mitogens, in part, through SP-A [51
], which also inhibits the Th2 immune response in vivo in a mouse model of Aspergillus fumigatus
-induced allergic disease [15
]. We found that expression of SP-R210 was markedly increased in T lymphocytes after stimulation by M. tuberculosis
() and therefore, hypothesize that this may provide a back-up control mechanism to dampen T cell activation, in addition to the immunosuppressive effects of SP-A mediated through alveolar macrophages. Activated T cells that express SP-R210 may become susceptible to the effects of SP-A, which can directly inhibit cell-cycle progression of T cells and attenuate intracellular calcium release in the absence of APC [19
]. Further studies are needed to clarify the direct effects of SP-A on T cells.
High levels of TGF-β1 are associated with active pulmonary tuberculosis [50
], but its precise role in the pathogenesis of tuberculosis remains unclear. TGF-β1 inhibits expression of SP-A and other SPs by alveolar type II epithelial cells [54
], and we recently found that SP-A levels were reduced during the first 15 days of pulmonary Mycobacterium bovis
BCG infection, preceding development of adaptive immunity [27
]. We speculate that SP-R210 mediates secretion of TGF-β1 by alveolar macrophages, which in turn inhibits SP-A production, facilitating initiation of protective Th1 immune response against tuberculosis. In later stages of infection, SP-R210-mediated TGF-β1 secretion may help control the inflammatory response and reduce tissue damage.
IL-10 is produced during the local immune response to M. tuberculosis
], but its role in tuberculosis pathogenesis is uncertain. Mice overexpressing IL-10 from T cells had increased susceptibility to reactivation tuberculosis at later stages of infection [57
]. On the other hand, IL-10-deficient mice did not have enhanced resistance to tuberculosis [58
]. The primary role of IL-10 in the lung is believed to be to reduce excessive inflammation and injury [59
]. Alveolar type I and type II epithelial cells, recruited monocytes, and alveolar macrophages are important sources of IL-10 in the lung [60
], and the latter three cell types are known to express SP-R210 and respond to SP-A [26
]. In combination with previous studies, our findings support the hypothesis that SP-R210 is involved in balancing immune responses to mycobacterial infection through secretion of IL-10 and TGF-β1 in vivo.
In conclusion, we found that in a physiologically relevant system, α-SP-R210n antibodies inhibited production of TNF-α and IFN-γ as well as proliferation of antigen-specific T lymphocytes through eliciting enhanced production of IL-10 and TGF-β1. These results provide insight into the receptor-mediated mechanisms that underlie the anti-inflammatory roles of SP-A in innate and adaptive immunity in multiple previous studies. The present experimental approach obviates technical limitations arising from using native or recombinant SP-A molecules that may be contaminated with different amounts of LPS [64
] or TGF-β1 [65
]. The agonistic α-SP-R210n antibodies represent a new tool to understand the physiological functions of the SP-A and its receptor SP-R210 in modulating innate and adaptive immune responses.