Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

Background

In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.

Methods

BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.

Results

AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4⁺ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.

Conclusion

Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.     

Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.      

Behavioural and physiological consequences of acute social defeat in growing gilts: effects of the social environment

Endocrine, behavioural and immunologic processes, together with body growth, were evaluated in gilts that were defeated at 10 weeks of age in resident-intruder tests. Immediately after defeat, gilts were either separated from or reunited with a familiar conspecific (litter-mate; always a barrow). Gilts were assigned to one of four treatments: (a) DI: defeat, followed by isolation (separation from original litter-mate; n=8); (b) I: no defeat, isolation (control group; n=9); (c) DP; defeat, followed by pair-housing (reunion with original litter-mate; n=8); and (d) P: no defeat, pair-housing (control group; n=8). The following general conclusions were derived: (1) social defeat caused pronounced short-term elevations in hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal medullary activities, and of prolactin levels. Moreover, as soon as 1h after defeat, percentages of blood lymphocytes and neutrophilic granulocytes were, respectively, decreased and increased; (2) social defeat had some long-lasting influence on behaviour and physiology, but isolation predominantly determined responses in the longer term. Defeat, as well as isolation, resulted in increased cardiovascular activities compared to P controls, as observed in a novel object test (NOT: +7 days) and an aversion test (AVT: +14 days). Moreover, defeated as well as isolated gilts did not habituate to a repeated novel environment test (NET: -7, +2 and +7 days) in terms of frequencies of vocalising, whereas P controls did. Isolation, through the separation from any other pig, was responsible for the other observed long-term characteristics, which developed progressively. Isolated gilts showed high mobilities and high cortisol responses in the repeated NET (+7 days), not being habituated. This contrasted the reactions of pair-housed gilts, which were much reduced. In addition to their high cardiovascular activities in the NOT and the AVT, isolated gilts also displayed higher heart rates in the repeated NET and during human presence following the NOT, compared to pair-housed gilts. Finally, isolated gilts were more inhibited to approach a novel object (in the NOT) than pair-housed pigs; and (3) stress responses of defeated gilts were modulated by the subsequent social environment. Stimulation of the HPA-axis (plasma- and salivary cortisol) was prolonged in those defeated gilts which were isolated (observed in the first hour). Changes in leucocyte subsets were still observed after 3 days in DI, but were 'normalised' within 1 day in DP gilts. Two days after defeat, habituation to the repeated NET in terms of mobility and salivary cortisol responses occurred in control and DP gilts, but not in DI gilts. We argue that these effects of the social environment shortly after defeat were related to a stress-reducing effect of a stable social relationship, i.e. social support.