Preoperative Immature Neutrophils Predict Clinical Outcomes in Patients with Uncomplicated Type-B Aortic Dissection After Thoracic Endovascular Aortic Repair.

Purpose: Inflammation is a hallmark of the initial development and progression of aortic dissection. This study aimed to investigate the predictive value of preoperative neutrophils in aorta-related adverse events (AAEs) after thoracic endovascular aortic repair (TEVAR) for type B aortic dissection (TBAD). Patients and Methods: A total of 80 patients with TBAD undergoing TEVAR were enrolled in our hospital. Preoperative inflammatory markers, including white blood cells (WBCs), neutrophils, neutrophil-to-lymphocyte ratio (NLR) and plasma high-sensitivity C-reactive protein (hs-CRP), were measured. Circulating neutrophil subpopulation was determined by flow cytometry. Kaplan-Meier curve was performed to determine whether neutrophil subsets independently predicted aorta-related adverse events (AAEs) after TEVAR. Results: Compared with control group, the prevalence of hypertension and the levels of inflammatory indicators including WBCs, total neutrophils, NLR, immature neutrophils and hs-CRP were significantly higher in TBAD patients. Receiver operating characteristic (ROC) curve showed that NLR, absolute number of total neutrophils and percent CD10- immature neutrophils had excellent area under curves. During the 18-month follow-up, 16 (20.0%) were reported to occur AAEs, while 4 deaths (5.0%) were documented. Percent immature neutrophil was markedly higher in TBAD patients experiencing AAEs as compared with those without AAEs. Kaplan-Meier curve and Cox regression analysis demonstrated that percent immature neutrophil was the only predictor correlated with the occurrence of AAEs (hazard ratio 7.66, 95% CI: 2.91, 20.17, P = 0.018). Conclusion: Increased CD10- immature neutrophils could act as a potential biomarker related to long-term adverse outcomes in TBAD patients following TEVAR.

Circulating neutrophil extracellular trap (NET)-forming 'rogue' neutrophil subset, immunotype [DEspR + CD11b +], mediate multi-organ failure in COVID-19-an observational study.

Background: Cumulative research show association of neutrophils and neutrophil extracellular traps (NETs) with poor outcomes in severe COVID-19. However, to date, there is no curative intent therapy able to block neutrophil/NETs-mediated progression of multi-organ dysfunction. Because of emerging neutrophil heterogeneity, the study of subsets of circulating NET-forming neutrophils [NET + Ns] as mediators of multi-organ failure progression among patients with COVID-19 is critical to identification of therapeutic targets. Methods: We conducted a prospective observational study of circulating levels of CD11b + [NET + N] immunotyped for dual endothelin-1/signal peptide receptor (DEspR ±) expression by quantitative immunofluorescence-cytology and causal mediation analysis. In 36 consented adults hospitalized with mod-severe COVID-19, May to September 2020, we measured acute multi-organ failure via SOFA-scores and respiratory failure via SaO2/FiO2 (SF)-ratio at time points t1 (average 5.5 days from ICU/hospital admission) and t2 (the day before ICU-discharge or death), and ICU-free days at day28 (ICUFD). Circulating absolute neutrophil counts (ANC) and [NET + N] subset-specific counts were measured at t1. Spearman correlation and causal mediation analyses were conducted. Results: Spearman correlation analyses showed correlations of t1-SOFA with t2-SOFA (rho r S = 0.80) and ICUFD (r S = -0.76); circulating DEspR + [NET + Ns] with t1-SOFA (r S = 0.71), t2-SOFA (r S = 0.62), and ICUFD (r S = -0.63), and ANC with t1-SOFA (r S = 0.71), and t2-SOFA (r S = 0.61).Causal mediation analysis identified DEspR + [NET + Ns] as mediator of 44.1% [95% CI:16.5,110.6] of the causal path between t1-SOFA (exposure) and t2-SOFA (outcome), with 46.9% [15.8,124.6] eliminated when DEspR + [NET + Ns] were theoretically reduced to zero. Concordantly, DEspR + [NET + Ns] mediated 47.1% [22.0,72.3%] of the t1-SOFA to ICUFD causal path, with 51.1% [22.8,80.4%] eliminated if DEspR + [NET + Ns] were reduced to zero. In patients with t1-SOFA > 1, the indirect effect of a hypothetical treatment eliminating DEspR + [NET + Ns] projected a reduction of t2-SOFA by 0.98 [0.29,2.06] points and ICUFD by 3.0 [0.85,7.09] days. In contrast, there was no significant mediation of SF-ratio through DEspR + [NET + Ns], and no significant mediation of SOFA-score through ANC. Conclusions: Despite equivalent correlations, DEspR + [NET + Ns], but not ANC, mediated progression of multi-organ failure in acute COVID-19, and its hypothetical reduction is projected to improve ICUFD. These translational findings warrant further studies of DEspR + [NET + Ns] as potential patient-stratifier and actionable therapeutic target for multi-organ failure in COVID-19. Supplementary Information: The online version contains supplementary material available at 10.1186/s41231-023-00143-x.

The Regulation of Neutrophil Migration in Patients with Sepsis: The Complexity of the Molecular Mechanisms and Their Modulation in Sepsis and the Heterogeneity of Sepsis Patients.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Common causes include gram-negative and gram-positive bacteria as well as fungi. Neutrophils are among the first cells to arrive at an infection site where they function as important effector cells of the innate immune system and as regulators of the host immune response. The regulation of neutrophil migration is therefore important both for the infection-directed host response and for the development of organ dysfunctions in sepsis. Downregulation of CXCR4/CXCL12 stimulates neutrophil migration from the bone marrow. This is followed by transmigration/extravasation across the endothelial cell barrier at the infection site; this process is directed by adhesion molecules and various chemotactic gradients created by chemotactic cytokines, lipid mediators, bacterial peptides, and peptides from damaged cells. These mechanisms of neutrophil migration are modulated by sepsis, leading to reduced neutrophil migration and even reversed migration that contributes to distant organ failure. The sepsis-induced modulation seems to differ between neutrophil subsets. Furthermore, sepsis patients should be regarded as heterogeneous because neutrophil migration will possibly be further modulated by the infecting microorganisms, antimicrobial treatment, patient age/frailty/sex, other diseases (e.g., hematological malignancies and stem cell transplantation), and the metabolic status. The present review describes molecular mechanisms involved in the regulation of neutrophil migration; how these mechanisms are altered during sepsis; and how bacteria/fungi, antimicrobial treatment, and aging/frailty/comorbidity influence the regulation of neutrophil migration.

Increased levels of low density neutrophils (LDNs) in myocardial infarction.

BACKGROUND: Recent evidence suggests that neutrophils are highly plastic cells that can display heterogeneous phenotypes. Low-density neutrophils (LDNs) have been described in many inflammatory conditions, and are thought to represent an immature, hyperactivated subtype of neutrophils. Neutrophils are significantly involved in the inflammatory response to myocardial infarction (MI), although we do not know the extent to which LDNs exist, or function, in MI. This study sought to determine the frequency and phenotype of LDNs in MI patients, compared to healthy subjects (HS). METHODS: LDNs and normal-density neutrophils (NDNs) were isolated from the peripheral blood of MI subjects (n = 12) and HSs (n = 12) using density gradient centrifugation. LDNs and NDNs were analysed by flow cytometry to identify neutrophils (CD66b+CD15+CD14-CD3-CD19- cells) and examine neutrophil activation (CD11b, CD66b and CD15) and maturity (CD33 and CD16). RESULTS: We identified LDNs within the peripheral blood mononuclear cell (PBMC) fraction of blood, and this population is significantly enriched in MI patients (1.04 ± 0.75% of PBMCs), compared to HS (0.29 ± 0.24%, p = .003). Across both cohorts, LDNs express significantly higher levels of CD66b and CD15, indicating a heightened state of activation compared to NDNs. In this study, LDNs were described as CD33highCD16low, compared to CD33lowCD16high NDNs, indicating the immaturity of this neutrophil subtype. CONCLUSIONS: An increase in the frequency of hyperactivated, immature LDNs is an immunological feature of MI. We highlight a potential pathological role of LDNs in MI, which underscores the need to expand our current understanding of this subtype in MI and other cardiovascular diseases (CVDs).

"Rogue" [DEspR+CD11b+] neutrophil subset correlates with severity in spontaneous intracerebral hemorrhage.

Objective: Cumulative clinical, cellular, and molecular evidence reinforces the role of neutrophils in secondary brain injury in spontaneous intracerebral hemorrhage (sICH). However, since generalized neutrophil inhibition is detrimental, identification of targetable "rogue" neutrophil subsets associated with sICH severity is key. Methods: In a pilot prospective observational study of consented patients with sICH, we immunotyped whole blood to assess circulating neutrophil markers (~day 3 after ICH symptoms onset): (a) DEspR±CD11b± neutrophils by flow cytometry, (b) DEspR±CD11b± neutrophil extracellular trap (NET)-forming neutrophils by immunofluorescence cytology, and (c) neutrophil-lymphocyte ratio (NLR). Using Spearman rank correlation (r) with Bonferroni correction, we assessed the association of neutrophil markers with same-day clinical and neuroimaging parameters of sICH severity, index ICH score, 90-day modified Rankin Scale (mRS) score, and potential interrelationships. As comparators, we assessed same-day plasma biomarkers elevated in sICH: interleukin-6/IL-6, myeloperoxidase/MPO, soluble-terminal complement complex/sC5b-9, endothelin-1/ET-1, and mitochondrial/nuclear DNA ratio (mt/nDNA ratio). Results: We detected strong correlations [r(n = 13) > 0.71, power > 0.8, Bonferroni corrected p B < 0.05] for all three neutrophil markers with 90-day mRS score, differentially for DEspR+CD11b+ neutrophil counts, and NLR with perihematomal edema (PHE) volume and for DEspR+CD11b+ NET-forming neutrophil counts with intraparenchymal hemorrhage (IPH)-volume. Only DEspR+CD11b+ neutrophil counts show a strong correlation with index ICH score, same-day Glasgow Coma Scale (GCS) score, and NLR and NET-forming neutrophil counts. The sum of the ICH score and three neutrophil markers exhibited the highest correlation: [r(n = 13) 0.94, p B = 10-5]. In contrast, plasma biomarkers tested were elevated except for MPO but exhibited no correlations in this pilot study. Conclusion: Strong correlation with multiple sICH severity measures, NET formation, and NLR identifies DEspR+CD11b+ neutrophils as a putative "rogue" neutrophil subset in sICH. The even stronger correlation of the sum of three neutrophil markers and the index ICH score with 90-day mRS outcome reinforces early neutrophil-mediated secondary brain injury as a key determinant of outcome in patients with sICH. Altogether, data provide a basis for the formal study of the DEspR+CD11b+ neutrophil subset as a potential actionable biomarker for neutrophil-driven secondary brain injury in sICH. Data also show ex vivo analysis of patients with sICH neutrophils as a translational milestone to refine hypotheses between preclinical and clinical studies.

Kinetics of Neutrophil Subsets in Acute, Subacute, and Chronic Inflammation.

At homeostasis the vast majority of neutrophils in the circulation expresses CD16 and CD62L within a narrow expression range, but this quickly changes in disease. Little is known regarding the changes in kinetics of neutrophils phenotypes in inflammatory conditions. During acute inflammation more heterogeneity was found, characterized by an increase in CD16dim banded neutrophils. These cells were probably released from the bone marrow (left shift). Acute inflammation induced by human experimental endotoxemia (LPS model) was additionally accompanied by an immediate increase in a CD62Llow neutrophil population, which was not as explicit after injury/trauma induced acute inflammation. The situation in sub-acute inflammation was more complex. CD62Llow neutrophils appeared in the peripheral blood several days (>3 days) after trauma with a peak after 10 days. A similar situation was found in the blood of COVID-19 patients returning from the ICU. Sorted CD16low and CD62Llow subsets from trauma and COVID-19 patients displayed the same nuclear characteristics as found after experimental endotoxemia. In diseases associated with chronic inflammation (stable COPD and treatment naive HIV) no increases in CD16low or CD62Llow neutrophils were found in the peripheral blood. All neutrophil subsets were present in the bone marrow during homeostasis. After LPS rechallenge, these subsets failed to appear in the circulation, but continued to be present in the bone marrow, suggesting the absence of recruitment signals. Because the subsets were reported to have different functionalities, these results on the kinetics of neutrophil subsets in a range of inflammatory conditions contribute to our understanding on the role of neutrophils in health and disease.

Instant intra-operative neutropenia despite the emergence of banded (CD16dim/CD62Lbright) neutrophils in peripheral blood - An observational study during extensive trauma-surgery in pigs.

INTRODUCTION: Deregulation of polymorphonuclear neutrophils (PMNs) is an essential step in the development of inflammatory complications upon trauma. Different neutrophil subtypes have been identified recently, however, the role of neutrophil subtypes in immunoregulation upon trauma is unclear. We hypothesize that extensive trauma surgery causes instant progressive heterogeneity of the blood neutrophil pool, and increased appearance of young (CD16dim/CD62Lbright) neutrophils in peripheral blood. MATERIAL AND METHODS: A standardized extensive thoraco-abdominal porcine trauma surgery model was utilized, and 12 animals were included. Blood was collected at defined timepoints and neutrophil numbers and subtypes were studied by flowcytometry. Neutrophil subtypes were identified by differences in cell surface expression levels of CD16 (FcγRIII) and CD62L (L-selectin). Porcine neutrophil subtypes were further characterized after flow sorting. RESULTS: Eleven animals survived the 3-hour surgical protocol. Neutrophil numbers dropped significantly from a mean of 8,6 ± 3,5 × 106 to 2,4 ± 1,8 × 106 cells/ml during 180 min, (p<0.001). Simultaneously, the blood PMN population became increasingly heterogeneous due to the appearance of new neutrophil subtypes. Cell sorting experiments and cytological analysis revealed that these porcine subtypes had specific morphological characteristics, mimicking their human counterparts. At baseline, 88% ± 1 percent of circulatory PMNs comprised of mature (CD16bright/CD62Lbright) PMNs, while at 3 h the blood PMN pool consisted of 59% ± 2 percent of mature subtypes (p<0.001). Despite a marked drop in neutrophil levels during surgery, absolute and relative numbers of banded (CD16dim/CD62Lbright) neutrophils continued to rise throughout surgery. CONCLUSION: Standardized extensive trauma surgery was associated with instant progressive neutropenia and increased heterogeneity of the blood neutrophil pool. Furthermore, three different neutrophil subsets in peripheral porcine blood were identified over the course of surgery. Further studies should clarify their precise role in the development of early organ failure upon extensive trauma surgery. This for the first time exemplifies experimentally the time constraints and impact of damage control surgery after severe trauma.

The Secretive Life of Neutrophils Revealed by Intravital Microscopy.

Neutrophils are the most abundant circulating leukocyte within the blood stream and for many years the dogma has been that these cells migrate rapidly into tissues in response to injury or infection, forming the first line of host defense. While it has previously been documented that neutrophils marginate within the vascular beds of the lung and liver and are present in large numbers within the parenchyma of tissues, such as spleen, lymph nodes, and bone marrow (BM), the function of these tissue resident neutrophils under homeostasis, in response to pathogen invasion or injury has only recently been explored, revealing the unexpected role of these cells as immunoregulators or immune helpers and also unraveling their heterogeneity and plasticity. Neutrophils are highly motile cells and the use of intravital microscopy (IVM) to image cells within their environment with little manipulation has dramatically increased our understanding of the function, migratory behavior, and interaction of these short-lived cells with other innate and adaptive immune cells. Contrary to previous dogma, these studies have shown that marginated and tissue resident neutrophils are the first responders to pathogens and injury, critical in limiting the spread of infection and contributing to the orchestration of the subsequent immune response. The interplay of neutrophils, with other neutrophils, leukocytes, and stroma cells can also modulate and tune their early and late response in order to eradicate pathogens, minimize tissue damage, and, in certain circumstances, contribute to tissue repair. In this review, we will follow the extraordinary journey of neutrophils from their origin in the BM to their death, exploring their role as tissue resident cells in the lung, spleen, lymph nodes, and skin and outlining the importance of neutrophil subsets, their functions under homeostasis, and in response to infection. Finally, we will comment on how understanding these processes in greater detail at a molecular level can lead to development of new therapeutics.

On the origin of low-density neutrophils.

Here we elaborate on the origin of low(er)-density neutrophils (LDNs) to better understand the variation found in literature. Supplemented with original data, we test the hypothesis that buoyant density of neutrophils is characterized by a spectrum that as a whole shifts to a lower density after activation. Both the 20% highest density (HDNs) and 20% lowest density (LDNs) neutrophils from healthy donors were isolated by Percoll of different densities. Using this method we found that LDNs were significantly better in T-cell suppression and bacterial containment than their 20% highest density counterparts. We found no statistically relevant differences in neutrophil survival or bacterial phagocytosis. Stimulation of healthy donor neutrophils with N-formyl-methionyl-leucyl-phenylalanine induced LDNs co-segregating with peripheral blood mononuclear cells after Ficoll separation. These in vitro induced LDNs showed increased activation markers compared to HDNs and were comparable to the activation markers found on the LDN fraction seen in patients with chronic inflammatory conditions such as present in cancer patients. This all fits with the hypothesis that the density of neutrophils is distributed in a spectrum partially coupled to maturation. Additionally a shift in this spectrum can be induced by in vitro stimulation or by activation in disease.

The olfactomedin-4 positive neutrophil has a role in murine intestinal ischemia/reperfusion injury.

Olfactomedin-4 (OLFM4) identifies a subset of neutrophils conserved in both mouse and man, associated with worse outcomes in several inflammatory conditions. We investigated the role of OLFM4-positive neutrophils in murine intestinal ischemia/reperfusion (IR) injury. Wild-type (WT) C57Bl/6 and OLFM4 null mice were subjected to intestinal IR injury and then monitored for survival or tissues harvested for further analyses. In vivo intestinal barrier function was determined via functional assay of permeability to FITC-dextran. OLFM4 null mice had a significant 7-d survival benefit and less intestinal barrier dysfunction compared with WT. Early after IR, WT mice had worse mucosal damage on histologic examination. Experiments involving adoptive transfer of bone marrow demonstrated that the mortality phenotype associated with OLFM4-positive neutrophils was transferrable to OLFM4 null mice. After IR injury, WT mice also had increased intestinal tissue activation of NFκB and expression of iNOS, 2 signaling pathways previously demonstrated to be involved in intestinal IR injury. In combination, these experiments show that OLFM4-positive neutrophils are centrally involved in the pathologic pathway leading to intestinal damage and mortality after IR injury. This may provide a therapeutic target for mitigation of intestinal IR injury in a variety of common clinical situations.-Levinsky, N. C., Mallela, J., Opoka, A., Harmon, K., Lewis, H. V., Zingarelli, B., Wong, H. R., Alder, M. N. The olfactomedin-4 positive neutrophil has a role in murine intestinal ischemia/reperfusion injury.

Neutrophils in Post-myocardial Infarction Inflammation: Damage vs. Resolution?

Inflammation not only plays a crucial role in acute ischemic cardiac injury, but also contributes to post-infarction repair and remodeling. Traditionally, neutrophils have been merely considered as detrimental in the setting of an acute myocardial infarction. However, recently published studies demonstrated that neutrophils might also play an important role in cardiac repair by regulating reparative processes. An emerging concept is that different neutrophil subsets exist, which might exhibit separate functional properties. In support of the existence of distinct neutrophil subsets in the ischemic heart, transcriptional changes in cardiac neutrophils have been reported within the first few days after myocardial infarction. In addition, there is an increasing awareness of sex-specific differences in many physiological and pathophysiological responses, including cardiovascular parameters and inflammation. Of particular interest in this context are recent experimental data dissecting sex-specific differences in neutrophil signaling after myocardial infarction. Unraveling the distinct and possibly stage-dependent properties of neutrophils in cardiac repair may provide new therapeutic strategies in order to improve the clinical outcome for myocardial infarction patients. This review will briefly discuss recent advances in our understanding of the neutrophil functional repertoire and emerging insights of sex-specific differences in post-myocardial infarction inflammatory responses.

Neutrophils From Children With Systemic Juvenile Idiopathic Arthritis Exhibit Persistent Proinflammatory Activation Despite Long-Standing Clinically Inactive Disease.

Background: Systemic juvenile idiopathic arthritis (SJIA) is a chronic childhood arthropathy with features of autoinflammation. Early inflammatory SJIA is associated with expansion and activation of neutrophils with a sepsis-like phenotype, but neutrophil phenotypes present in longstanding and clinically inactive disease (CID) are unknown. The objective of this study was to examine activated neutrophil subsets, S100 alarmin release, and gene expression signatures in children with a spectrum of SJIA disease activity. Methods: Highly-purified neutrophils were isolated using a two-step procedure of density-gradient centrifugation followed by magnetic-bead based negative selection prior to flow cytometry or cell culture to quantify S100 protein release. Whole transcriptome gene expression profiles were compared in neutrophils from children with both active SJIA and CID. Results: Patients with SJIA and active systemic features demonstrated a higher proportion of CD16+CD62Llo neutrophil population compared to controls. This neutrophil subset was not seen in patients with CID or patients with active arthritis not exhibiting systemic features. Using imaging flow cytometry, CD16+CD62Llo neutrophils from patients with active SJIA and features of macrophage activation syndrome (MAS) had increased nuclear hypersegmentation compared to CD16+CD62L+ neutrophils. Serum levels of S100A8/A9 and S100A12 were strongly correlated with peripheral blood neutrophil counts. Neutrophils from active SJIA patients did not show enhanced resting S100 protein release; however, regardless of disease activity, neutrophils from SJIA patients did show enhanced S100A8/A9 release upon PMA stimulation compared to control neutrophils. Furthermore, whole transcriptome analysis of highly purified neutrophils from children with active SJIA identified 214 differentially expressed genes (DEG) compared to neutrophils from healthy controls. The most significantly upregulated gene pathway was Immune System Process, including AIM2, IL18RAP, and NLRC4. Interestingly, this gene set showed intermediate levels of expression in neutrophils from patients with long-standing CID yet persistent serum IL-18 elevation. Indeed, all patient samples regardless of disease activity demonstrated elevated inflammatory gene expression, including inflammasome components and S100A8. Conclusion: We identify features of neutrophil activation in SJIA patients with both active disease and CID, including a proinflammatory gene expression signature, reflecting persistent innate immune activation. Taken together, these studies expand understanding of neutrophil function in chronic autoinflammatory disorders such as SJIA.

Neutrophil, quo vadis?

Neutrophils were traditionally considered to be a homogeneous population of terminally differentiated cells with very defined roles in inflammation and fighting infections. However, recent advances in neutrophil research challenge this limited view and demonstrate that neutrophils are highly versatile, play different roles in various pathologic scenarios, and are heterogeneous. With this, it is becoming clear that one term-"neutrophil"-is too general, and more precise nomenclature is urgently required. In this mini review, we discuss the knowns and unknowns in neutrophil terminology and highlight the critical questions that should be addressed for the establishment of clear neutrophil nomenclature.

NET-producing CD16high CD62Ldim neutrophils migrate to tumor sites and predict improved survival in patients with HNSCC.

The concept of functional neutrophil subsets is new and their clinical significance in malignancies is unknown. Our study investigated the role of CD16dim CD62Lhigh , CD16high CD62Lhigh and CD16high CD62Ldim neutrophil subsets in head and neck squamous cell carcinoma (HNSCC) patients. These neutrophil subsets may play different roles in immune-related activity in cancer, based on their profile, activation state and migration ability within a tumor site, which may be important in predicting cancer prognoses. Tumor biopsies and blood were obtained from newly diagnosed untreated HNSCC patients and healthy controls. Neutrophil subsets and their phenotype were characterized using flow cytometry. Isolated granulocytes were assessed for anti-tumor immune functions. Compared to controls HNSCC patients exhibited increased CD16high CD62Ldim neutrophils in blood; this subset displayed a distinct phenotypes with high expression of CD11b and CD18. This subset was prone to migrate into the tumor facilitated by tumor-derived IL-8. Furthermore, IL-8 was also found to activate neutrophils and thereby promoting subset transition. Various assays demonstrated that activated CD16high CD62Ldim neutrophils inhibited migration, proliferation and induced apoptosis of FaDu cancer cells. Neutrophil elastase detected in activated CD16high CD62Ldim neutrophils and tumor biopsies suggested that CD16high CD62Ldim neutrophils impart anti-tumoral activity via neutrophil extracellular traps. Furthermore, increased fraction of CD16high CD62Ldim neutrophils was shown to correlate with an increased survival rate. Our study demonstrates the clinical relevance of the CD16high CD62Ldim neutrophil subset, providing evidence for its increased migration capacity, its anti-tumor activity including increased NET formation and finally its correlation with increased survival in HNSCC patients.

Elevated mean neutrophil volume represents altered neutrophil composition and reflects damage after myocardial infarction.

Myocardial infarction (MI) induces an inflammatory response in which neutrophils fulfill a prominent role. Mean neutrophil volume (MNV) represents the average size of the circulating neutrophil population. Our goal was to determine the effect of MI on MNV and investigate the mechanisms behind MNV elevation. MNV of 84 MI patients was compared with the MNV of 209 stable angina patients and correlated to simultaneously measured CK levels. Fourteen pigs were subjected to temporary coronary balloon occlusion and blood was sampled at multiple time points to measure MNV. Echocardiography was performed followed by ex vivo infarct size assessment after 72 h. MNV was higher in MI patients compared to stable angina patients (602 SD26 AU vs. 580 SD20 AU, p < 0.0001) and correlated with simultaneously measured CK levels (R = 0.357, p < 0.0001). In pigs, MNV was elevated post-MI (451 SD11 AU vs. 469 SD12 AU), p < 0.0001). MNV correlated with infarct size (R = 0.705, p = 0.007) and inversely correlated with left ventricular ejection fraction (R = -0.718, p = 0.009). Cell sorting revealed an increased presence of banded neutrophils after MI, which have a higher MNV compared to mature neutrophils post-MI (495 SD14 AU vs. 478 SD11 AU, p = 0.012). MNV from coronary sinus blood was higher than MNV of neutrophils from simultaneously sampled arterial blood (463 SD7.6 AU vs. 461 SD8.6 AU, p = 0.013) post-MI. The current study shows MNV is elevated and reflects cardiac damage post-MI. MNV increases due to altered neutrophil composition and systemic neutrophil activation. MNV may be an interesting parameter for prognostic assessment in MI and provide new insights into pathological innate immune responses evoked by ischemia-reperfusion.

Plasma levels of OLFM4 in normals and patients with gastrointestinal cancer.

Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 µg/ml, mean 0.028 µg/ml while 10% of both normals and patients with cancers had OLFM4 between 4 and 60 µg/ml, mean 15 µg/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers.

Olfactomedin-4 autoantibodies give unusual c-ANCA staining patterns with reactivity to a subpopulation of neutrophils.

Testing for the presence of ANCAs in circulation is part of the clinical examinations routinely performed upon suspected autoimmune disorders, mainly vasculitis. The autoantibodies are typically directed toward neutrophil MPO or PR3. These are major granule-localized proteins, and similar to all hitherto-described ANCA antigens, they are expressed by all neutrophils, and ANCA-containing sera thus give rise to uniform reactivity toward all neutrophils in a sample. In this paper, we describe sera from 2 unrelated patients with diffuse inflammatory symptoms that gave rise to peculiar c-ANCA patterns, only reacting with a subpopulation (roughly 30%) of human neutrophils. By immunoblotting, both sera reacted to the same antigen, which was expressed in intracellular granules. The antigen could be released to the extracellular milieu through secretion but also through the formation of NETs. Neutrophils have long been considered a homogenous cell population, but it is becoming increasingly clear that distinct subpopulations, defined by the presence or absence of certain proteins, exist. One such marker that defines a neutrophil subset is the granule protein OLFM4. The unusual, subset-restricted c-ANCA sera reacted only with OLFM4-positive neutrophils, and MS analysis revealed that the autoantigen was, in fact, OLFM4. These data describe for the first time a c-ANCA pattern reactive to only a subpopulation of neutrophils and identify the granule protein OLFM4 as a novel autoantigen.