Lena Gunnarsson

Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils

Proteinase 3 (PR3) is found in granules of all neutrophils but also on the plasma membrane of a subset of neutrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of neutrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where all mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3‐ and CD177‐expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener’s granulomatosis and polycythemia vera.

A defence of the category ‘women’

Against influential strands of feminist theory, I argue that there is nothing essentialist or homogenising about the category ‘women’. I show that both intersectional claims that it is impossible to separate out the ‘woman part’ of women, and deconstructionist contentions that the category ‘women’ is a fiction, rest on untenable meta-theoretical assumptions. I posit that a more fruitful way of approaching this disputed category is to treat it as an abstraction. Drawing on the philosophical framework of critical realism I elucidate the nature of the vital and inevitable process of abstraction, as a means of finding a way out of the theoretical and methodological impasse that the ‘ban’ on the category ‘women’ has caused. Contrary to many contemporary feminist theorists, I contend that, although the category ‘women’ does not reflect the whole reality of concrete and particular women, it nevertheless refers to something real, namely the structural position as woman.

Elevated neutrophil membrane expression of proteinase 3 is dependent upon CD177 expression

Proteinase 3 (PR3) is a major autoantigen in anti‐neutrophil cytoplasmic antibodies (ANCA)‐associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double‐positive or double‐negative for mPR3 and CD177. The proportion of double‐positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony‐stimulating factor and granulocyte–macrophage colony‐stimulating factor. Pro‐PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double‐positive cells. In cells sorted based on membrane expression, CD177–mRNA was several‐fold higher in mPR3+ cells. When exogenous PR3 was added to CD177‐transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.

Decreased Neutrophil Apoptosis in Quiescent ANCA-Associated Systemic Vasculitis

Background ANCA-Associated Systemic Vasculitis (AASV) is characterized by leukocytoclasis, accumulation of unscavenged apoptotic and necrotic neutrophils in perivascular tissues. Dysregulation of neutrophil cell death may contribute directly to the pathogenesis of AASV. Methods Neutrophils from Healthy Blood Donors (HBD), patients with AASV most in complete remission, Polycythemia Vera (PV), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and renal transplant recipients (TP) were incubated in vitro, and the rate of spontaneous apoptosis was measured by FACS. Plasma levels of cytokines and sFAS were measured with cytometric bead array and ELISA. Expression of pro/anti-apoptotic factors, transcription factors C/EBP-α, C/EBP-β and PU.1 and inhibitors of survival/JAK2-pathway were measured by real-time-PCR. Results AASV, PV and RA neutrophils had a significantly lower rate of apoptosis compared to HBD neutrophils (AASV 50±14% vs. HBD 64±11%, p<0.0001). In RA but not in AASV and PV, low apoptosis rate correlated with increased plasma levels of GM-CSF and high mRNA levels of anti-apoptotic factors Bcl-2A1 and Mcl-1. AASV patients had normal levels of G-CSF, GM-CSF and IL-3. Both C/EBP-α, C/EBP-β were significantly higher in neutrophils from AASV patients than HBD. Levels of sFAS were significantly higher in AASV compared to HBD. Conclusion Neutrophil apoptosis rates in vitro are decreased in AASV, RA and PV but mechanisms seem to differ. Increased mRNA levels of granulopoiesis-associated transcription factors and increased levels of sFAS in plasma were observed in AASV. Additional studies are required to define the mechanisms behind the decreased apoptosis rates, and possible connections with accumulation of dying neutrophils in regions of vascular lesions in AASV patients.

Increased neutrophil membrane expression and plasma level of proteinase 3 in systemic vasculitis are not a consequence of the - 564 A/G promotor polymorphism.

Several findings link proteinase 3 (PR3) to small vessel vasculitis. Besides being a major target of anti‐neutrophil cytoplasm antibodies (ANCA), previous findings have shown increased circulating levels of PR3 in vasculitis patients, increased levels of neutrophil membrane‐PR3 (mPR3) expression and a skewed distribution of the − 564 A/G polymorphism in the promotor region of the PR3 gene. In this study we elucidate how these three findings relate to each other. The plasma concentration of PR3 was measured by enzyme‐linked immunosorbent assay (ELISA), mPR3 expression by fluorescence activated cell sorter (FACS) and the gene polymorphism by real‐time polymerase chain reaction (PCR). We compared results from 63 patients with ANCA‐associated systemic vasculitis (AASV) with 107 healthy blood donors. In accordance with previous reports, AASV patients had increased plasma concentrations of PR3 compared to healthy controls (mean 224 µg/l versus 155 µg/l, P < 0·0001). They also showed an increased number of mPR3‐positive neutrophils (60%versus 42%, P < 0·001). However, contrary to a previous report, we found no skewed distribution of the polymorphism in PR3 gene. There was a weak correlation between mPR3 mean fluorescence intensity (MFI) and plasma PR3 among healthy controls and myeloperoxidase–ANCA (MPO–ANCA)‐positive patients (r = 0·24, P = 0·015 and r = 0·52, P = 0·011, respectively). In conclusion, increased plasma PR3 and high expression of mPR3 are associated with small vessel vasculitis, but neither of them is a consequence of the − 564 A/G polymorphism of the PR3 gene promotor.

The naturalistic turn in feminist theory : a Marxist-realist contribution

After a time dominated by nature-phobia, a naturalistic turn is emerging within feminist theory. Welcoming this new theoretical embrace of nature and sympathising with its insistence that nature is not feminism’s enemy, this article nevertheless points to some problematic features of this turn. Focusing on Elizabeth Grosz’s postmodernist readings of Charles Darwin, I suggest that their emphasis of nature’s dynamic, indeterminate and enabling qualities both implies a politically unmotivated glorification of the dynamic and unruly, and as such obscures the important fact that nature also works as a constraining factor on societies. I demonstrate, from the point of view of a Marxist-realist perspective, why an acknowledgement of nature’s limiting force is crucial for the coherence of any theoretical account of the workings of social systems. The article also addresses the feminist imperative to transcend the dualism between nature and culture, and shows how the concept of emergence offers a solution to dilemmas that tend to appear in connection to such efforts of transcendence.

Epitope mapping of anti-PR3 antibodies using chimeric human/mouse PR3 recombinant proteins

Autoantibodies against proteinase 3 (PR3) and myeloperoxidase (MPO) (ANCA = anti‐neutrophil cytoplasmic antibodies) are used as diagnostic tools for patients with small vessel vasculitis. ANCA are detected by different assays, but the correlation between the results of these assays is generally poor. The overall aim of the study was to provide a framework for the future development of new assays with an increased diagnostic yield. In order to express discrete epitopes of human PR3 (hPR3), the nonantigenic molecules murine PR3 (mPR3) and human leucocyte elastase (HLE) were used as a framework. We constructed recombinant chimeric vectors and were able to produce 6 hPR3/mPR3 proteins and 3 hPR3/HLE proteins. Anti‐PR3 monoclonal antibodies differed in their binding pattern to the chimeras, but no distinct binding region could be identified for any monoclonal antibody. The recombinant hPR3/mPR3 were also tested in ELISA with sera from patients with Wegeners granulomatosis with renal involvement. The results show that patients have antibodies to different constructs, indicating that the patients vary in their antibody repertoire from the beginning of the disease, and that patients may have antibodies from a broad range of clones early in the course of the disease. Recombinant hPR3/mPR3 chimeric proteins have a potential to be used as antigens in future ANCA assays.

Neutrophils from vasculitis patients exhibit an increased propensity for activation by anti-neutrophil cytoplasmic antibodies.

Anti‐neutrophil cytoplasmic antibodies (ANCA) are thought to be pathogenic in ANCA‐associated vasculitis (AAV) by stimulating polymorphonuclear leucocytes (PMNs) to degranulate and produce reactive oxygen species (ROS). The aim of this study was to investigate if PMNs from AAV patients are stimulated more readily by ANCA compared with PMNs from healthy controls (HCs). Differences in ANCA characteristics that can account for different stimulation potential were also studied. PMNs from five AAV patients and five HCs were stimulated with 10 different immunoglobulins (Ig)Gs, purified from PR3–ANCA‐positive patients, and ROS production, degranulation and neutrophil extracellular trap (NET) formation was measured. ANCA levels, affinity and clinical data of the AAV donors were recorded. The results show that PMNs from AAV patients produce more intracellular ROS (P = 0·019), but degranulate to a similar extent as PMNs from HCs. ROS production correlated with NET formation. Factors that may influence the ability of ANCA to activate PMNs include affinity and specificity for N‐terminal epitopes. In conclusion, our results indicate that PMNs from AAV patients in remission behave quite similarly to HC PMNs, with the exception of a greater intracellular ROS production. This could contribute to more extensive NET formation and thus an increased exposure of the ANCA autoantigens to the immune system.

Why we keep separating the ‘inseparable’: Dialecticizing intersectionality

Disputes about how to understand intersectional relations often pivot around the tension between separateness and inseparability, where some scholars emphasize the need to separate between different intersectional categories while others claim they are inseparable. In this article the author takes issue with the either/or thinking that underpins an unnecessary and unproductive polarization in the debate over the in/separability of intersectional categories. Drawing on Roy Bhaskar’s dialectical critical realist philosophy, the author argues that we can think of intersectional categories as well as different ontological levels as both distinct and unified and elaborates on the issue of how significance of the dialectical notion of unity-in-difference for intersectional studies. As part of the argument the author addresses the issue of what it actually means for something to be distinct or separate as opposed to inseparable or unified with something else, demonstrating that lack of clarity about this is at the heart of polarized arguments about separateness versus inseparability in intersectionality theory.

Epitope shift of proteinase-3 anti-neutrophil cytoplasmic antibodies in patients with small vessel vasculitis.

Anti‐neutrophil cytoplasmic antibodies against proteinase 3 (PR3‐ANCA) are used as diagnostic tools for patients with small vessel vasculitis (AASV). We have produced chimeric mouse/human PR3 molecules and investigate changes in reactivity over time and the possible relationship between epitope specificity and clinical course. Thirty‐eight PR3‐ANCA‐positive patients diagnosed between 1990 and 2003 were followed until December 2005. Plasma was collected at each out‐patient visit and older samples were retrieved retrospectively. Patients reacted with multiple epitopes at the time of diagnosis. At subsequent relapses 12 patients shifted reactivity, in 11 cases from epitopes located in the C‐terminal towards epitopes in the N‐terminal. Patients with reactivity against N‐terminal parts of PR3 at diagnosis had a significantly lower relapse rate, 30% compared to 78% in the group with predominantly C‐terminal reactivity (P = 0·04). The reactivity pattern did not correlate to outcome measured as death, end‐stage renal disease or vasculitis activity index score (VDI) at 5 years. Further research is necessary to conclude if this is a general phenomenon.