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Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5.

ABSTRACT: Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.

SUBMITTER: Ponda MP

PROVIDER: S-EPMC5111683 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5.

Ponda Manish P MP Breslow Jan L JL

Proceedings of the National Academy of Sciences of the United States of America 20161024 45

Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purifi ...[more]

PMID: 27791187

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Project description:BACKGROUND:Schistosomes are blood dwelling parasitic worms that cause the debilitating disease schistosomiasis. Here we examined the influence of the parasites on their external environment by monitoring the impact of adult Schistosoma mansoni worms on the murine plasma proteome in vitro and, in particular, on how the worms affect the blood coagulation protein high molecular weight kininogen (HK). METHODS:Following the incubation of adult schistosomes in murine plasma, two-dimensional differential in-gel electrophoresis (2D-DIGE) was conducted to look for changes in the plasma proteome compared with control plasma. A major change to the blood protein kininogen (HK) was observed, and the interaction of Schistosoma mansoni parasite with this protein alone was then investigated by western blot analysis and activity assays. Finally, the generation of bradykinin from HK was monitored using a bradykinin detection kit. RESULTS:The most striking change to the plasma proteome concerned HK; while the full-length protein was more abundant in control plasma, carboxyl-terminal truncated forms were more abundant in plasma that contained schistosomes. Incubating parasites in buffer with pure HK followed by Western blot analysis confirmed that human HK is degraded by the worms. The resulting digestion pattern differed from that brought about by kallikrein, a host serine protease that normally acts on HK to release the vasodilator bradykinin. We found that live schistosomes, while digesting HK, do not generate bradykinin nor do they cleave a chromogenic kallikrein substrate. Since the cleavage of HK by the worms is not impeded by the serine protease inhibitor PMSF but is blocked by the cysteine protease inhibitor E64c, we hypothesize that schistosome tegumental cysteine proteases are responsible for HK cleavage. CONCLUSIONS:Since proteomic and biochemical studies have revealed that the schistosome tegument contains two cysteine proteases belonging to the calpain family (SmCalp1 and SmCalp2) we conclude that these are likely responsible for the HK cleavage reported here. Schistosome cleavage of HK should help impede blood clotting and inflammation around the worms in vivo and so promote their ease of movement within the vasculature of their hosts.

Project description:The purpose of this study was use molecular imaging targeting coagulation pathway and inflammation to better understand the pathophysiology of silent brain ischemia (SBI) and monitor the effects of factor XIIa inhibition.SBI can be observed in patients who undergo invasive vascular procedures. Unlike acute stroke, the diffuse nature of SBI and its less tangible clinical symptoms make this disease difficult to diagnose and treat.We induced SBI in mice by intra-arterial injection of fluorescently labeled microbeads or fractionated clot into the carotid artery. After SBI induction, diffusion-weighted magnetic resonance imaging was performed to confirm the presence of microinfarcts in asymptomatic mice. Molecular imaging targeting the downstream factor XIII activity (single-photon emission computed tomography/computed tomography) at 3 h and myeloperoxidase activity (magnetic resonance imaging) on day 3 after SBI induction were performed, without and with the intravenous administration of a recombinant selective factor XIIa inhibitor derived from the hematophagous insect Triatoma infestans (rHA-Infestin-4). Statistical comparisons between 2 groups were evaluated by the Student t test or Mann-Whitney U test.In SBI-induced mice, we found abnormal activation of the coagulation cascade (factor XIII activity) and increased inflammation (myeloperoxidase activity) close to where emboli lodge in the brain. rHA-Infestin-4 administration significantly reduced ischemic damage (53% to 85% reduction of infarct volume, p < 0.05) and pathological coagulation (35% to 39% reduction of factor XIII activity, p < 0.05) without increasing hemorrhagic frequency. Myeloperoxidase activity, when normalized to the infarct volume, did not significantly change with rHA-Infestin-4 treatment, suggesting that this treatment does not further decrease inflammation other than that resulting from the reduction in infarct volume.Focal intracerebral clotting and inflammatory activity are part of the pathophysiology underlying SBI. Inhibiting factor XIIa with rHA-Infestin-4 may present a safe and effective treatment to decrease the morbidity of SBI.

Project description:RationaleStudies identified kininogen as a potential biomarker of preeclampsia, a major cause of adverse maternal outcomes. High-molecular-weight kininogen (HK) and its activated form participate in numerous pathways associated with establishing and maintaining pregnancy. However, dynamic changes in HK and naturally occurring HK-derived peptides during the natural course of pregnancy are largely unknown.MethodsLongitudinal serum samples during the course of normal pregnancy (trimesters T1, T2, T3) from 60 pregnant women were analyzed by western blot with an anti-HK antibody. Circulating peptides in longitudinal serum specimens derived from 50 participants were enriched using nanoporous silica thin films. Peptides were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and database searching. Relative quantification was performed using MaxQuant and in-house scripts. Normality was evaluated by either ANOVA or Friedman tests with p < 0.05 for statistical significance.ResultsWestern blotting revealed that HK significantly decreased during normal pregnancy (T1 vs T2, p < 0.05; T1 vs T3, p < 0.0001). A 100 kDa intermediate increased during pregnancy (T1 vs T2, p < 0.005; T1 vs T3, p < 0.01). Moreover, the heavy chain (T1 vs T2, p < 0.0001; T1 vs T3, p < 0.0001; T2 vs T3, p < 0.01) and light chain (T1 vs T2, p < 0.0001; T1 vs T3, p < 0.0001; T2 vs T3, p < 0.05) significantly increased during pregnancy. LC/MS/MS analysis identified 180 kininogen-1 peptides, of which 167 mapped to domain 5 (D5). Seventy-three peptides with ten or more complete data sets were included for further analysis. Seventy peptides mapped to D5, and 3, 24, and 43 peptides showed significant decrease, no trend, and significant increase, respectively, during pregnancy.ConclusionsThis study demonstrates dynamic changes in HK and naturally occurring HK-derived peptides during pregnancy. Our study sheds light on the gestational changes of HK and its peptides for further validation of them as potential biomarkers for pregnancy-related complications.

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Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5.

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Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5.

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