Neutrophil activation in systemic capillary leak syndrome (Clarkson disease)

Abstract Systemic capillary leak syndrome (SCLS; Clarkson disease) is a rare orphan disorder characterized by transient yet recurrent episodes of hypotension and peripheral oedema due to diffuse vascular leakage of fluids and proteins into soft tissues. Humoral mediators, cellular responses and genetic features accounting for the clinical phenotype of SCLS are virtually unknown. Here, we searched for factors altered in acute SCLS plasma relative to matched convalescent samples using multiplexed aptamer‐based proteomic screening. Relative amounts of 612 proteins were changed greater than twofold and 81 proteins were changed at least threefold. Among the most enriched proteins in acute SCLS plasma were neutrophil granule components including bactericidal permeability inducing protein, myeloperoxidase and matrix metalloproteinase 8. Neutrophils isolated from blood of subjects with SCLS or healthy controls responded similarly to routine pro‐inflammatory mediators. However, acute SCLS sera activated neutrophils relative to remission sera. Activated neutrophil supernatants increased permeability of endothelial cells from both controls and SCLS subjects equivalently. Our results suggest systemic neutrophil degranulation during SCLS acute flares, which may contribute to the clinical manifestations of acute vascular leak.

Specific aetiological factors and pathways contributing the profound vascular barrier breakdown characteristic of acute SCLS flares are unknown. Although more than 80% of SCLS patients have a monoclonal gammopathy of unknown significance, there is no direct or indirect evidence that the monoclonal IgG has any role in disease pathogenesis. 2 In our studies of more than 65 patients with a confirmed diagnosis of SCLS, we found that several inflammatory cytokines (eg TNFα, CCL2, CXCL10) and mediators of vascular permeability (VEGF, Angpt-2) were significantly elevated in sera from patients during acute episodes compared to convalescent intervals. 2,3 Treatment of normal vascular endothelial cells (ECs) with acute but not remission sera from these patients disrupted vascular integrity through mechanisms including internalization of VE-cadherin and actin stress fibre formation.
In order to broaden our search for mediators unique to SCLS and/ or uncover a signature of humoral factors characteristic of acute attacks, we used the slow-off rate modified aptamer (SOMA)scan platform, an aptamer-based highly multiplexed proteomic assay capable of detecting more than 1300 protein analytes simultaneously across the whole dynamic range from only a small volume of plasma with high sensitivity and specificity. 4 The SOMAscan approach uncovered more than 600 proteins, whose relative amounts were increased more than twofold in acute plasma relative to matched baseline plasma from the same patients. Most notably, several neutrophil granule proteins were profoundly elevated in acute SCLS plasma. Acute but not remission sera from SCLS patients activated neutrophils, and supernatants from activated neutrophils induced hyperpermeability of ECs. These results suggest that activated and/or degranulated neutrophils contribute to the pathogenesis of acute SCLS.

| Subjects
Patients were diagnosed with SCLS according to established criteria, specifically a history of one or more transient episodes of hypotension, elevated haematocrit and hypoalbuminemia. 5,6 Patients were seen at the Clinical Center of the National Institutes of Health. Written informed consent was obtained from each patient and the study protocol plasma and sera were collected and stored from both remission and active disease intervals where available. Neutrophils were available only from asymptomatic patients. Anonymized age-, sex-and race-matched blood from healthy volunteers were obtained from the NIH Blood Bank for neutrophil isolation and used as controls.

| SOMAscan and ELISA
Nine pairs of EDTA-plasma samples obtained at or near the onset of an acute SCLS crisis and from the same patient during a convalescent interval were analysed using the SOMAscan Assay Kit for human plasma 1.3k V3.2 (SomaLogic, Inc, cat.#900-00011) according to the standard protocol for EDTA plasma from the manufacturer's and as described previously. 8

| Neutrophil isolation
Neutrophils were isolated from whole blood using Hypaque Ficoll separation. Briefly, 1:1 PBS diluted blood was added onto the top of Ficoll (lymphocyte separation medium, MP Biomedicals), centrifuged at 800x g at room temperature for 20 minutes with the brake off. Neutrophils were retrieved from the bottom layer containing RBCs. After RBC lysis using ACK lysing buffer, cell pellets were washed twice in PBS. Neutrophil purity was routinely ~95% based on surface marker expression (CD16 + , CD3 − /CD19 − ) as determined by flow cytometry.

| Neutrophil activation/degranulation
For analysis of neutrophil activation, freshly isolated neutrophils were incubated with buffer or stimuli in culture medium at 37°C for the indicated time periods, followed by termination of the reaction by addition of ice-cold PBS. Cells were then stained with Live/Dead violet

| Endothelial permeability measurements
An electric cell-substrate impedance sensing (ECIS) assay was used to assess endothelial barrier function. Electrical resistance was measured across endothelial monolayers at 4,000 Hz using the ECIS Zθ apparatus (Applied BioPhysics) as described previously. 2 Briefly, ECs were serum starved in endothelial basal medium (EBM2, Lonza) plus 0.2% BSA for 5 hours followed by addition of test reagents.
Resistance was recorded over a period of 20 hours. Each condition was measured in duplicate in a single experiment and averaged.
Absolute resistance values were normalized by subtracting the resistance at time zero (pre-treatment); the maximal change in resistance was calculated as percentage change over time zero.

| Statistical analysis
ELISA and flow cytometric data were analysed with GraphPad Prism 7 software package. Data were analysed by two-way ANOVA with multiple corrections testing, and non-parametric tests were used for flow data analysis (Mann-Whitney test for analysis of two groups; Kruskal-Wallis for analysis of multiple groups). P < 0.05 were considered significant.

| Proteomic profile of acute SCLS plasma
Although the clinical symptoms of acute SCLS crises, including hemodynamic collapse and anasarca, are quite dramatic, patients are typically asymptomatic in between episodes. Although SCLS flares may occur in the absence of obvious triggers, recent survey studies suggest that infections (typically upper respiratory) precede SCLS attacks in 35%-50% of adult patients. 1,9 These findings suggest that infection-related inflammatory mediators may induce vascular leakage in some patients. Indeed, in limited prior screening of acute SCLS sera, we found elevated levels of pro-inflammatory mediators including TNFα, CXCL10, CCL2 and IL-8. 2,3 To more fully probe the SCLS proteome, we performed SOMAscan profiling of 1305 proteins in nine matched plasma samples obtained by venipuncture during acute or convalescent intervals. The characteristics of these patients are described in Table 1. This assay identified 612 proteins, whose relative amounts were changed at least twofold in episodic plasma relative to baseline (P < 0.05) (full list in Table S1), and all but three of them were increased. Eighty-one proteins were increased at least threefold in episodic plasma (P < 0.001) ( Figure 1A). With one exception, the protein profile of episodic and baseline samples clustered with one another. Consistent with previous studies of our entire SCLS cohort, several cytokines and vascular permeability mediators including CCL2, CXCL10, IL-12, IL-1β, TNFα, adrenomedullin, Angpt-2 and VEGFA were significantly elevated in episodic plasma compared to remission plasma (Table S2). 2,3, 10 We also discovered enrichment of several proteins not previously associated with SCLS including surfactant protein D S(P-D), capping actin protein (CAPG), chymase, allograft inflammatory factor (AIF1), the TNFα superfamily member LIGHT and the stressorin IL-16 (Table 2). We analysed functional enrichment using Ingenuity pathway analysis. In the category of "Disease & Biological Function", "Inflammatory response" was the statistically most significant category (P = 10 −15 ) ( Figure 1B); "Upstream Regulators" enriched in acute SCLS samples included several pro-inflammatory mediators such as LPS, IL-1β and TNFα (all P < 10 −6 ) ( Figure 1C). Finally, the top "Canonical Pathways" implicated in acute SCLS were IL-8 and glucocorticoid signalling (P = 10 −6 ) ( Figure 1D).

| Neutrophil activation signature in SCLS disease flares
Among the proteins that were increased the most in acute SCLS plasma were neutrophil granule components bactericidal permeability increasing protein (BPI) (19.5-fold), matrix metalloproteinase 8 (MMP8, 7.9-fold) and MMP9 (4.5-fold) ( Table 3). As noted above functionally enriched pathways in acute samples included signalling involving IL-8, a well-known neutrophil chemoattractant, and LPS, which also activates neutrophils. 11 Given these findings, we  Figure S1). Thus, we conclude that hemoconcentration alone is unlikely to account for the increased MPO in acute SCLS plasma relative to convalescence. Instead, our findings suggested that widespread neutrophil degranulation occurs in circulation during SCLS crises.

| Intrinsic neutrophil function in SCLS
We next determined whether neutrophils from SCLS patients are intrinsically hyper-responsive to inflammatory stimuli, which could account for the increased granule components in acute plasma. We  subset, compared to cells treated with matched remission sera.

| Acute SCLS sera induce neutrophil activation
( Figure 4A-D). This result suggests that SCLS serum components constrained to acute disease flares activate neutrophils, which in turn could account for the increase in granule components in acute SCLS plasma relative to that from asymptomatic individuals.

| Neutrophil degranulation and vascular barrier integrity
We tested the effects of neutrophil granule contents on endothelial barrier function. First, we stimulated neutrophils isolated from healthy donors with medium alone or fMLP. We confirmed neutrophil degranulation by MPO assay ( Figure S3A). We then treated ECs with supernatants from the activated neutrophils and measured transendothelial electrical resistance (TER) across monolayers in real time.
fMLP-stimulated neutrophil supernatants from two separate donors the resistance of cells exposed to medium alone remained relatively constant over a period of 10 hours ( Figure S3B). We next determined the effects of degranulated neutrophil supernatants on ECs isolated and expanded from peripheral blood of SCLS patients or healthy controls BOECs. 7 The activated neutrophil supernatants reduced TER in a pattern similar to commercially supplied HMVECs ( Figure 5A,B).
However, the maximal decrease in resistance in BOECs from SCLS subjects and controls was equivalent at both early (30 minutes) and later (15 hours) time points. Thus, although neutrophil granule constituents exert permeability-inducing effects on ECs, our data do not suggest hypersensitivity of SCLS ECs to this stimulus.  Another key finding is the increased activation of neutrophils induced by acute but not basal sera, as indicated by the increased percentage of the cells shedding CD62L. A CD16 bright /CD62L dim neutrophil subset appears to be quite distinct from other neutrophil subpopulations in vivo; published work has suggested that such cells may be recruited from bone marrow to circulation in response to acute inflammation. 13 This subset is less adherent to ECs and may play an immunosuppressive function as it preferentially migrates to splenic white pulp and inhibits T cell proliferation. 12,21 Whether this neutrophil subpopulation has a distinct function in SCLS or simply reflects de novo activation requires further study.

| D ISCUSS I ON
In summary, through multiplexed proteomic screening of plasma, we have discerned a panoply of proteins enriched in plasma during F I G U R E 6 SOMAscan and other discovery-driven approaches to unraveling systemic capillary leak syndrome (SCLS) disease mechanisms. Analysis of shared genes encoding proteins increased greater than twofold in acute vs remission SCLS plasma and transcripts significantly enriched >log2-fold in SCLS Bloodoutgrowth endothelial cells vs controls using GeneVenn. Shared genes listed at bottom SCLS flares that suggests widespread neutrophil activation/degranulation. Our results demonstrate the utility of SOMAscan as a discovery tool for disease mechanisms. A comparison of genes encoding proteins elevated in acute SCLS plasma with transcripts overexpressed SCLS ECs compared to controls (unpublished RNA-Seq data) revealed a possible cellular source for several SCLS-enriched proteins ( Figure 6). As but one example, platelet-derived growth factor B, which is secreted by ECs, stimulates proliferation and activation of pericytes, which are smooth muscle-like cells surrounding microvessels. Pericyte coverage is required for maintenance of microvascular endothelial barrier function in acute inflammation. 22 We believe that these and other discovery-driven approaches will eventually lead to advances in classification of SCLS and development of biomarkers for treatment efficacy.

This research was supported in part by the Intramural Research
Program, NIAID/NIH.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflicts of interest.

AUTH O R S H I P CO NTR I B UTI O N S
XZ, TAL, DBK, XG, HHO performed experiments and analysed data, and edited the paper. XZ, JIG, SMP and KMD designed and oversaw the project, and wrote and edited the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.