Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Duke University Medical Center

Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A-deficient condition. Wild-type (WT) and SP-A null (SP-A(-/-)) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A(-/-) mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A(-/-) mice. LPS-treated SP-A(-/-) mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A(-/-) mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.

Nitric oxide mediates relative airway hyporesponsiveness to lipopolysaccharide in surfactant protein A-deficient mice.

Although many studies have shown that pulmonary surfactant protein (SP)-A functions in innate immunity, fewer studies have addressed its role in adaptive immunity and allergic hypersensitivity. We hypothesized that SP-A modulates the phenotype and prevalence of dendritic cells (DCs) and CD4(+) T cells to inhibit Th2-associated inflammatory indices associated with allergen-induced inflammation. In an OVA model of allergic hypersensitivity, SP-A(-/-) mice had greater eosinophilia, Th2-associated cytokine levels, and IgE levels compared with wild-type counterparts. Although both OVA-exposed groups had similar proportions of CD86(+) DCs and Foxp3(+) T regulatory cells, the SP-A(-/-) mice had elevated proportions of CD4(+) activated and effector memory T cells in their lungs compared with wild-type mice. Ex vivo recall stimulation of CD4(+) T cell pools demonstrated that cells from the SP-A(-/-) OVA mice had the greatest proliferative and IL-4-producing capacity, and this capability was attenuated with exogenous SP-A treatment. Additionally, tracking proliferation in vivo demonstrated that CD4(+) activated and effector memory T cells expanded to the greatest extent in the lungs of SP-A(-/-) OVA mice. Taken together, our data suggested that SP-A influences the prevalence, types, and functions of CD4(+) T cells in the lungs during allergic inflammation and that SP deficiency modifies the severity of inflammation in allergic hypersensitivity conditions like asthma.

Lung effector memory and activated CD4+ T cells display enhanced proliferation in surfactant protein A-deficient mice during allergen-mediated inflammation.

Pulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation.

Surfactant protein A integrates activation signal strength to differentially modulate T cell proliferation.

TCR signaling plays a critical role in regulatory T cell (Treg) development. However, the mechanism for tissue-specific induction of Tregs in the periphery remains unclear. We observed that surfactant protein A (SP-A)-deficient mice have impaired expression of Foxp3 and fewer CD25(+)Foxp3(+) Tregs after ex vivo stimulation and after stimulation with LPS in vivo. The addition of exogenous SP-A completely reversed this phenotype. Although SP-A is known to inhibit T cell proliferation under certain activation conditions, both IL-2 levels as well as active TGF-β levels increase on extended culture with exogenous SP-A, providing a key mechanism for the maintenance and induction of Tregs. In addition, kinetic suppression assays demonstrate that SP-A enhances the frequency of functional Foxp3(+) Tregs in responder T cell populations in a TGF-β-dependent manner. In mice treated with LPS in vivo, Tregs increased ∼160% in wild-type mice compared with only a 50% increase in LPS-treated SP-A(-/-) mice 8 d after exposure. Taken together, these findings support the hypothesis that SP-A affects T cell immune function by the induction of Tregs during activation.

Surfactant protein A modulates induction of regulatory T cells via TGF-β.

Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A-deficient mice on a C57BL/6 background (H2b; SP-A(-/-)) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A-deficient recipient mice that have undergone an allogeneic BMT [SP-A(-/-)alloBMT] or SP-A-sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A-deficient recipient mice that have undergone a syngeneic BMT or SP-A-sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A(-/-) alloBMT or SP-A-sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A(-/-)alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A(-/-)alloBMT mice also had increased colon expression of IL-1β, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.

Novel role for surfactant protein A in gastrointestinal graft-versus-host disease.

Caveolin-1, the hallmark protein of caveolae, is highly expressed within the lung in the epithelium, endothelium, and in immune cells. In addition to its classical roles in cholesterol metabolism and endocytosis, caveolin-1 has also been shown to be important in inflammatory signaling pathways. In particular, caveolin-1 is known to associate with the nitric oxide synthase enzymes, downregulating their activity. Endotoxins, which are are composed mainly of lipopolysaccharide (LPS), are found ubiquitously in the environment and can lead to the development of airway inflammation and increased airway hyperresponsiveness (AHR).

Charles Giamberardino

Department of Pediatrics

Duke University Medical Center

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Charles Giamberardino

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Duke University Medical Center

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Department of Pediatrics