NF-κB/MAPK activation underlies ACVR1-mediated inflammation in human heterotopic ossification.

BACKGROUND: Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear. METHODS: We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-ß activated kinase 1 (TAK1), and NF-κB pathways. RESULTS: FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-ß production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages. CONCLUSIONS: Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.

Immune responses to self-antigens in asthma patients: clinical and immunopathological implications.

Asthma leads to chronic airway inflammation that shares pathological features of chronic rejection after lung transplantation. Due to the significant role of autoimmunity in chronic rejection, we hypothesized that immunity to self-antigens may also be present in asthma. The goal was to define immune responses to self-antigens in patients with asthma. Blood and clinical data were collected from 99 asthmatics and 60 controls. Serum was analyzed for antibodies (Abs) to collagen V (ColV) by enzyme-linked immunosorbent assay and correlated with disease severity. Asthmatics' sera were tested in a human protein array to determine immune responses to other self-antigens. Asthmatics had higher concentrations of Abs to ColV (predominantly immunoglobulin G isotype) compared with controls (p < 0.01). These Abs correlated with severe asthma (p < 0.01) and corticosteroid use (p = 0.032). Additionally, Abs to novel self-antigens epidermal group factor receptor (EGFr), activin A type 1 receptor, and α-catenin were detected in asthmatics. We conclude that Abs to self-antigens (ColV, EGFr, activin A type 1 receptor, and α-catenin) are present in the sera of asthmatics, correlating with clinical disease. Epithelial damage from airway inflammation during asthma may result in the exposure of cryptic self-antigens or their determinants, resulting in immune response to self-antigens, which may contribute to the pathogenesis of asthma.

Platelets possess functional TGF-beta receptors and Smad2 protein.

TGF-beta1 plays a main role in tissue repair by regulating extracellular matrix production and tissue granulation. Platelets are one of the main sources of this cytokine in the circulation. The aim of this study was to evaluate the presence of the TGF-beta receptors on platelets, the effect of TGF-beta1 on platelet aggregation and the underlying intracellular mechanisms. TGF-beta receptors on platelets were studied by flow cytometry and their mRNA by PCR. Platelet aggregation was assessed by turbidimetric methods and intracellular pathways by Western blot. TGF-beta receptor type II and mRNA codifying for TbetaRI and TbetaRII were found in platelets. We demonstrated that TGF-beta1 did not trigger platelet aggregation by itself but had a modulating effect on ADP-induced platelet aggregation. Either inhibition or increase in platelet aggregation, depending on the exposure time to TGF-beta1 and the ADP concentration used, were shown. We found that platelets possess Smad2 protein and that its phosphorylation state is increased after exposure to TGF-beta1. Besides, TGF-beta1 modified the pattern of ADP-induced tyrosine phosphorylation. Increased phosphorylation levels of 64-, 80- and 125-kDa proteins during short time incubation with TGF-beta1 and increased phosphorylation of 64- and 125-kDa proteins after longer incubation were observed. The modulating effect of TGF-beta1 on platelet aggregation could play a role during pathological states in which circulating TGF-beta1 levels are increased and intravascular platelet activation is present, such as myeloproliferative disorders. In vascular injury, in which platelet activation followed by granule release generates high local ADP concentrations, it could function as a physiological mechanism of platelet activation control.

[Mutation of the activin receptor-like kinase 1(ALK1) gene and the expression of plasma thrombomodulin in type-2 hereditary hemorrhagic telangiectasia: a study of a Chinese family].

OBJECTIVE: To analysis and define the clinical phenotype and related mutation of hereditary hemorrhagic telangiectasia. (HHT). METHODS: The proband of a Chinese HHT family, female, aged 48, and 2 of her family members: her father, aged 74, and her brother, aged 44 underwent nasal cavity endoscopy and automatic photochonography to observe the capillaries in nasal mucosa. Samples of peripheral blood were collected. The exons 3, 7, and 8 of the activin receptor-like kinase 1 (ALK1) gene of the proband and her family members and 2 healthy people as contrds were amplified by polymerase chain reaction, and the PCR products were sequenced. The levels of plasma thrombomodulin (TM) of the proband and her family members were detected by Western blotting combined with density screening. RESULTS: Except the mother and sister-in-law, other 4 of the family members had epistaxis or bleeding in other sites. The proband and her father had obvious telangiectasis of nasal mucosa or finger skin respectively. ALK1 gene analysis confirmed that a C1231T mutation (CGG-->TGG) in the exon 8 existed in the proband, her brother, and her father, no mutation was found in her sister in law, her nephew, and the healthy persons. Six bands were shown in the expression of plasma thrombomodulin. Density screening was conducted for the 2 bands with differential expression: those with the molecular masses of 56,000 and 28,000. The result of density screening showed that the average value of screening at the 56,000 band was 218.3 in the normal controls, significantly higher than that of the HTT patients (145.1); and the average value of screening at the 28,000 band was 222.0 in the normal controls, significantly higher than that in the HTT patients (145.1). CONCLUSION: ALK1 gene mutation, a C1231T variation on exon 8, exists in Chinese type 2 HHT individuals. The levels of plasma thrombomodulin levels at the 56,000 and 28,000 fragments of the HTT patients are lower than of normal subjects whose significance and mechanism remain to be elucidated.