Granzymes A and B are serine-proteinases stored in the granules of activated cytotoxic T-lymphocytes and natural killer (NK) cells. Expression of granzymes in tissues can be used as an activation marker for cytotoxic cells. Using mAbs specific for human granzyme A or B in immunohistochemical staining techniques we investigated expression of granzyme A and B by lymphocytes infiltrating acutely rejected renal allografts. Twelve core needle biopsies were taken from ten different patients during an episode of acute rejection. Eleven biopsies contained high numbers of granzyme A and B positive lymphocytes infiltrating tubular epithelium, and vascular and glomerular structures. In one patient infiltrating lymphocytes did not express granzyme A and only low amounts of granzyme B. No correlation was found between the number of granzyme positive cells and the severity of the rejection as classified by conventional histological criteria. In one tissue specimen from a patient with a renal allograft without signs of rejection, the number of granzyme positive cells was much lower compared to that of the transplant group. In spite of the presence of a marked inflammatory infiltrate, no granzyme positive cells were detected in renal biopsies from patients with various inflammatory, not transplant-related, renal diseases. Phenotypic analysis showed that granzymes A and B were expressed by CD56+ NK cells and CD3+ cells, representing cytotoxic T-lymphocytes. Thus, this study demonstrates that granzyme A and B protein-expressing lymphocytes infiltrate the kidney allografts during an acute cellular rejection but not in several other inflammatory renal diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

The human serine proteases granzymes A and B are expressed in cytotoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo.

Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide-linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases.

Mycobacterium chimaera, a non-tuberculous mycobacterium, was recently identified as causative agent of deep-seated infections in patients who had previously undergone open-chest cardiac surgery. Outbreak investigations suggested an aerosol-borne pathogen transmission originating from water contained in heater-cooler units (HCUs) used during cardiac surgery. Similar thermoregulatory devices are used for extracorporeal membrane oxygenation (ECMO) and M. chimaera might also be detectable in ECMO treatment settings. We performed a prospective microbiological study investigating the occurrence of M. chimaera in water from ECMO systems and in environmental samples, and a retrospective clinical review of possible ECMO-related mycobacterial infections among patients in a pneumological intensive care unit. We detected M. chimaera in 9 of 18 water samples from 10 different thermoregulatory ECMO devices; no mycobacteria were found in the nine room air samples and other environmental samples. Among 118 ECMO patients, 76 had bronchial specimens analysed for mycobacteria and M. chimaera was found in three individuals without signs of mycobacterial infection at the time of sampling. We conclude that M. chimaera can be detected in water samples from ECMO-associated thermoregulatory devices and might potentially pose patients at risk of infection. Further research is warranted to elucidate the clinical significance of M. chimaera in ECMO treatment settings.

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• pDCs functionally express the IL-21 receptor and produce granzyme B in response to IL-21.• IL-21-induced granzyme B in pDC impairs pDC's capacity to induce T-cell proliferation.Plasmacytoid dendritic cells (pDCs) play a crucial role during innate immunity by secreting bulk amounts of type I interferons (IFNs) in response to Toll-like receptor (TLR)-mediated pathogen recognition. In addition, pDCs can also contribute to adaptive immunity by activation of antigen-specific T cells. Furthermore, it is well established that pDCs contribute to the pathogenesis of autoimmune diseases, including lupus. Interleukin-21 (IL-21) is a cytokine produced by activated CD4 1 T and natural killer T (NKT) cells and has a pleiotropic role in immunity by controlling myeloid DC-, NKT-, T-, and B-cell functions. It has remained elusive whether IL-21 affects pDCs. Here we investigate the role of IL-21 in human pDC activation and function and observe that IL-21 activates signal transducer and activator of transcription 3 in line with the finding that pDCs express the IL-21 receptor. Although IL-21 did not affect TLR-induced type I IFNs, IL-6, and TNF-a nor expression of major-histocompatibility-complex class II or costimulatory molecules, IL-21 markedly increased expression of the serine protease granzyme B (GrB). We demonstrate that GrB induction was, in part, responsible for IL-21-mediated downmodulation of CD4 1 T-cell proliferation induced by TLR preactivated pDCs. Collectively, our data provide evidence that pDCs are important cells to consider when investigating the role of IL-21 in immunity or pathogenesis. (Blood. 2013;121(16):3103-3111)

SummaryIt has been shown that the most important inhibitor of plasmin is α2-antiplasmin, however, other protease inhibitors are able to inhibit this proteolytic enzyme as well. The contribution of the various protease inhibitors to the inhibition of plasmin in vivo has never been quantitatively assessed.To assess the relative contribution of the different protease inhibitors on the inhibition of plasmin we developed a series of sensitive immunoassays for the detection of complexes between plasmin and the protease inhibitors α2-antiplasmin, α2-macroglobulin, antithrombin III, α1antitrypsin and C1-inhibitor, utilizing monoclonal antibodies that are specifically directed against complexed protease inhibitors and a monoclonal antibody against plasmin.It was confirmed that α2-antiplasmin is the most important inhibitor of plasmin in vivo, however, complexes of plasmin with α2-macroglobulin, antithrombin III, α1antitrypsin- and C1-inhibitor were also detected. Particularly during activation of fibrinolysis complexes between plasmin and inhibitors other than α2-antiplasmin were detected. It was observed that during different situations the inhibition profile of plasmin was not constant e.g. in patients with diffuse intravascular coagulation plasma levels of plasmin-α1-antitrypsin and plasmin-C1-inhibitor were increased whereas in plasma from patients who were treated with thrombolytic agents complexes of plasmin with α2-macroglobulin and with antithrombin III were significantly elevated.In conclusion, we confirmed the important role of α2-antiplasmin in the inhibition of plasmin, however, in situations in which fibrinolysis is activated other protease inhibitors also account for the inhibition of plasmin in vivo. Further investigations to assess the role of the various protease inhibitors in the fibrinolytic system can be assisted by the assays described in this study.

The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)-injury. MBL-associated serine protease (MASP)-2 appears to be crucial in this process. The serpin C1-inhibitor is the major inhibitor of MASP-2. In addition, aprotinin, a Kunitz-type inhibitor, was shown to inhibit MASP-2 activity in vitro.In this study we investigated whether the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP-2. Ex vivo LP was induced and detected by C4-deposition on mannan-coated plates. The MASP-2 activity was measured in a fluid-phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI-domains contribute to MASP-2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP-2, without affecting MASP-1 or the classical pathway proteases C1s and C1r. Kunitz-2 domain of TFPI is required for the inhibition of MASP-2. Considering the role of MASP-2 in complement-mediated I/R-injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.Keywords: Complement-coagulation crosstalk r Complement inhibition r MASP-2 r TFPI Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe complement system comprises over 30 different plasma proteins. The complement system consists of three activation pathways, each differing in their recognition mechanism while Correspondence: Mischa P. Keizer e-mail: m.keizer@sanquin.nl converging in a common terminal pathway at the level of complement component 3 (C3). The antibody-dependent classical pathway (CP) initiates the complement system via the binding of C1q to antibodies. Binding to specific sugar motifs by mannan-binding lectins (MBLs) or one of the ficolins activates the lectin pathway (LP) by subsequent activation of the MBL-associated serine proteases (MASPs). The alternative pathway activates spontaneously on surfaces that lack complement regulatory proteins and acts as amplification route for the other two pathways.www.eji-journal.eu Eur. J. Immunol. 2015. 45: 544-550 Innate immunity 545Besides the LP role as first line of defense against invading pathogens, by opsonization and induction of an inflammatory response, recent studies have shown that the LP also plays a prominent role in ischemia/reperfusion (I/R) injury. MBL-deficiency appears to be protective in human myocardial infarction [1], gastrointestinal I/R injury [1], and stroke [2]. This detrimental role of the LP in I/R injury has been confirmed in mice by selectively targeting MASP-2. MASP-2 knockout mice were protected from both gastrointestinal and myocardial I/R injury [3]. Together, these data indicate an important role for MASP-2 in aggravating cell damage in ischemi...