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The prognostic significance of eosinophilia after myeloablative allogeneic stem cell transplantation (ASCT) remains to be established. Patients, whom developed chronic graft-versus-host disease (cGVHD) after ASCT, were included ( n = 142). Eosinophil count was analyzed at cGVHD onset. We observed no significant association between EO and the grade of cGVHD, thrombocytopenia, nor extensive skin involvement. Importantly, we observed no significant association between cGVHD with concomitant eosinophilia and long-term clinical outcomes, and subgroup analyses revealed a considerable confounding effect of ongoing steroid treatment. In conclusion, we advocate that prognostic conclusions regarding cGVHD with concomitant eosinophilia after ASCT should be interpreted with caution.

The t(6;9)(p22;q34) chromosomal translocation is found in a subset of patients with acute myeloid leukemia (AML). The translocation results in a fusion between the nuclear phosphoprotein DEK and the nucleoporin NUP214 (previously CAN). The mechanism by which the fusion protein DEK-NUP214 contributes to leukemia development has not been identified, and disruptions of normal cellular functions by DEK-NUP214 have previously not been described. In the present study, a novel effect of the DEK-NUP214 fusion protein is demonstrated. Our findings reveal a substantial increase in global protein synthesis in DEK-NUP214 expressing cells. Furthermore, we conclude that this effect is not the result of dysregulated transcription but merely due to increased translation. Consistent with the association with AML, the increased protein synthesis mediated by DEK-NUP214 is restricted to cells of the myeloid lineage. Analysis of potential mechanisms for regulating protein synthesis shows that expression of DEK-NUP214 correlates to the phosphorylation of the translation initiation protein, EIF4E. The present data provide evidence that increase of translational activity constitutes a mechanism by which the leukemogenic effect of DEK-NUP124 may be mediated.

Background: Allogeneic haematopoietic stem cell transplantation (HSCT) is a procedure with inherent complications and intensive care may be necessary. We evaluated the short- and long-term outcomes of the HSCT recipients requiring admission to the intensive care unit (ICU). Methods: We retrospectively examined the outcome of 54 adult haematological HSCT recipients admitted to the ICU at the University Hospital Rigshospitalet between January 2007 and March 2012. Results: The overall in-ICU, in-hospital, 6-month and 1-year mortality rates were 46.3, 75.9, 79.6 and 86.5%, respectively. Mechanical ventilation had a statistically significant effect on in-ICU (p = 0.02), 6-month (p = 0.049) and 1-year (p = 0.014) mortality. Renal replacement therapy also had a statistically significant effect on in-hospital (p = 0.038) and 6-month (p = 0.026) mortality. Short ICU admissions, i.e. Conclusion: The poor prognosis of critically ill HSCT recipients admitted to the ICU was confirmed in our study. Mechanical ventilation, renal replacement therapy and an ICU admission of ≥10 days were each risk factors for mortality in the first year after ICU admission.

A 54-year-old RhD-negative male with del(20q)-positive myelodysplastic syndrome was transplanted with bone marrow from an HLA-identical RhD-positive sibling donor. Cytogenetic relapse was detected 21 months after stem cell transplantation (SCT), with reappearance of the original del(20q)-positive clone and reversion to recipient RhD-negative blood group. The patient received sequential donor lymphocyte infusions (DLIs), resulting in mild graft-versus-host disease and pure red cell aplasia. At 2 years post DLI, the patient remains in a stable condition, despite a dominance of recipient-derived erythro- and granulopoiesis originating in del(20q)-carrying progenitor cells. We conclude that reappearance of autologous erythropoiesis, upon relapse after allogeneic SCT, may be predictive of erythropenia after DLI and that re-emerging autologous del(20q)-positive erythropoiesis post DLI can provide a normal peripheral red blood cell count. Furthermore, in patients relapsing after blood-group-mismatched transplantation, a possible reversion to recipient blood group should be considered prior to blood transfusion or DLI.

The biosynthesis and processing of the neutrophil granule membrane protein CD63, present in azurophil granules, was investigated in four myeloid cell lines. The amount of CD63 synthesised differed, so did the amount of protein processed to high molecular weight forms, with the demonstration of a more prominent synthesis of CD63 in K562 cells. Newly synthesised CD63 was initially detected as two precursor forms with molecular weight of 32 and 35 kDa, respectively. These two initial forms were processed further to yield high molecular weight forms of CD63 with a mean molecular weight of 50 kDa. Treatment with endoglycosidase H or N-glycosidase F revealed a protein core, free from asparagine-linked carbohydrates, with a molecular weight of 23 kDa. Newly synthesised CD63 was susceptible to digestion with endoglycosidase H, and the protein was not completely resistant to endoglycosidase H until after 4 h of chase, indicating that transport through the medial and trans-Golgi complex with conversion of high-mannose carbohydrates to complex oligosaccharide side chains had occurred. This finding indicates a relatively long processing time for CD63 compared to that of soluble azurophil granule proteins. By digestion with O-glycanase, the existence of O-linked oligosaccharides on CD63 could not be demonstrated. Biosynthetic labelling of cells in the presence of brefeldin A showed the importance of a functional Golgi apparatus for processing of the protein to its high molecular weight forms.

Azurocidin is a multifunctional endotoxin- binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of azurocidin, cDNA encoding human preproazurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proazurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino- terminal processing enzymes. Translocation of azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproazurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human azurocidin. J. Leukoc. Biol. 66: 634–643; 1999.

The serine protease cathepsin G is synthesized during the promyelomonocytic stage of neutrophil and monocyte differentiation. After processing, including removal of an amino-terminal propeptide from the catalytically inactive proform, the active protease acquires a mature conformation and is stored in azurophil granules. To investigate the importance of the proform-conformation for targeting to granules, a cDNA encoding a double-mutant form of human preprocathepsin G lacking functional catalytic site and amino-terminal prodipeptide (CatG/Gly201/▵Gly19Glu20) was constructed, because we were not able to stably express a mutant lacking only the propeptide. Transfection of the cDNA to the rat basophilic leukemia RBL-1 and the murine myeloblast-like 32D cl3 cell lines resulted in stable, protein-expressing clones. In contrast to wild-type proenzyme, CatG/Gly201/▵Gly19Glu20 adopted a mature conformation cotranslationally, as judged by the early acquisition of affinity to the serine protease inhibitor aprotinin, appearing before the carboxyl-terminal processing and also in the presence of the Golgi-disrupting agent brefeldin A. The presence of a mature amino-terminus was confirmed by amino-terminal radiosequencing. As with wild-type proenzyme, CatG/Gly201/▵Gly19Glu20 was proteolytically processed carboxyl-terminally and glycosylated with asparagine-linked carbohydrates that were converted into complex forms. Furthermore, it was targeted to granules, as determined by subcellular fractionation. Our results show that the initial proform-conformation is not critical for intracellular sorting of human cathepsin G. Moreover, we demonstrate that double-mutant cathepsin G can achieve a mature conformation before carboxyl-terminal processing of the proform.© 1998 by The American Society of Hematology.

The neutral protease cathepsin G belongs to a family of hematopoietic serine proteases stored in the azurophil granules of the neutrophil granulocyte. To investigate the function of asparagine-linked carbohydrates in neutrophil serine proteases, we constructed a mutant cDNA, coding for human cathepsin G deficient of a functional glycosylation site, for use in a transgenic cellular model. Wild type and mutant cDNA were stably expressed in the rat basophilic/mast cell line RBL and in the murine myeloblast-like cell line 32D. Biosynthetic labeling, followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography, showed that carbohydrate-deficient cathepsin G was synthesized as a 29-kDa proform in both cell lines. The proform was proteolytically processed into a stable form with an apparent molecular mass of 27.5 kDa, indicating removal of the carboxyl-terminal prodomain. The mutant cathepsin G was enzymatically activated as determined by acquisition of affinity to aprotinin, a serine protease inhibitor. As for wild type cathepsin G, small amounts of the unprocessed form of the mutated enzyme were released from the cells, while the major part was transferred to a granular compartment as demonstrated by subcellular fractionation. Thus, neither processing leading to enzymatic activation nor granular sorting was obviously affected by the lack of oligosaccharides on the mutant cathepsin G. Our results therefore indicate that glycosylation is not essential for these processes. In addition to the previously utilized cell line RBL, we propose the 32D cell line as a suitable cellular model for transgenic expression of human neutrophil serine proteases.

The effects of brefeldin A, monensin, and the weak base NH4C1 on the biosynthesis and processing of cathepsin G and neutrophil elastase of myeloid cells were investigated. Monoblast-like U-937 cells were biosynthetically labeled with [35S]methionine, followed by subcellular fractionation, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophore-sis and fluorography. Brefeldin A inhibited proteolytic processing, intracellular transport, and secretion. The effects were reversible inasmuch as removal of brefeldin A resulted in a normal pattern of processing and transfer to high-density organelles, corresponding to lysosomes, and restitution of constitutive secretion of precursor forms. Both cathepsin G and neutrophil elastase acquired resistance to endoglycosidase-H, suggesting that conversion to complex oligosaccharide side chains also occurs in the presence of brefeldin A. Monensin and NH4Cl inhibited final proteolytic processing, indicating either that acidification is necessary for directing cathepsin G and neutrophil elastase to lysosomal-like organelles or that the protease(s) responsible for processing requires an acid pH. We conclude that pH-dependent proteolytic processing of cathepsin G and neutrophil elastase occurs in post-Golgi structures and that transfer to lysosomes or an immediately prelysosomal compartment is mandatory for complete processing. J. Leukoc. Biol. 55: 50–57; 1994.

In 1997-2003, a protocol for treatment of acute myeloid leukaemia (AML) (except promyelocytic leukaemia) was activated in four Swedish health care regions covering 50% of the national population. Based on cytogenetics and clinical findings, patients aged 18-60 yr were assigned to one of three risk groups. In this report we account for the long-term clinical outcome of enrolled patients. Patients received idarubicin and cytarabine in standard doses as induction therapy and consolidation courses included high-dose cytarabine. Allogeneic stem cell transplantation (allo-SCT) from an human leucocyte antigen-identical sibling was recommended in standard and poor-risk patients, whereas unrelated donor transplant was reserved for poor-risk patients. Autologous (auto-SCT) was optional for standard or poor risk patients not eligible for allo-SCT. Two hundred seventy-nine patients with de novo or secondary (9%) AML, median age 51 (18-60) yr, corresponding to 77% of all patients in the population, were included. Twenty (7%) patients were assigned to the good risk group, whereas 150 (54%) and 109 patients (39%) were assigned to standard- and poor-risk groups, respectively. Induction failures accounted for 55 patients; 16 early deaths eight of whom had white blood cell (WBC) >100 at diagnosis, and 39 refractory disease. Thus, complete remission (CR) rate was 80%. At study closure, the median follow-up time of living patients was 90 months. Median survival time from diagnosis in the whole group was 27 months and 4-yr overall survival (OS) rate was 44%. In good, standard, and poor risk groups, 4-yr OS rates were 60, 57 and 24%, respectively. Median relapse-free survival (RFS) time in CR1 was 25 months and RFS at 4 yr was 44%. Four-year RFS rates were significantly (P < 0.001) different between the three risk groups; 64% in good risk, 51% in standard risk and 27% in poor risk patients. One hundred-ten transplantations were performed in CR1; 74 allo-SCT (50 sibling, 24 unrelated donor), and 36 auto-SCT. Non-relapse mortality was 16% for allo-SCT patients. Outcome after relapse was poor with median time to death 163 d and 4-yr survival rate 17%. Three conclusions were: (i) these data reflect treatment results in a minimally selected population-based cohort of adult AML patients

The processing of the neutral proteases cathepsin G and neutrophil elastase, normally synthesized in myeloid precursor cells and stored in azurophil granules, were investigated by biosynthetic labeling with 14C- leucine of the monoblastic cell line U-937. The proteases were precipitated with specific antibodies and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. The transfer to lysosomes of newly synthesized proteases was demonstrated in pulse-chase labeling experiments followed by centrifugation of cell homogenates in a Percoll gradient. The presence of a closely spaced polypeptide band-doublet at intermediate gradient density suggested cleavage of the specific aminoterminal pro dipeptide extension before storage in lysosomes. The molecular heterogeneity observed for cathepsin G and neutrophil elastase seemed to be due to modifications occurring after sorting into lysosomes, most likely because of C-terminal processing. Modifications of the secreted enzymes were not detectable by SDS-PAGE. In contrast to other lysosomal enzymes, no phosphorylation was demonstrated. Newly synthesized cathepsin G and neutrophil elastase rapidly became resistant to endoglycosidase H, indicating transport through the medial and trans cisternae of the Golgi complex and conversion to “complex” oligosaccharide side chains. This conversion was inhibited by an agent swainsonine, but translocation from the Golgi complex and secretion were unaffected. The processing described may play a role in activation of the proteases.

Effective T-cell depletion is a prerequisite for haploidentical peripheral blood progenitor cell (PBPC) transplantation. This study was performed to investigate the performance of magnetic cell sorting-based direct large-scale T-cell depletion, which is an attractive alternative to standard PBPC enrichment procedures.PBPCs were harvested from 11 human leukocyte antigen (HLA)-haploidentical donors. T cells labeled with anti-CD3-coated beads were depleted with a commercially available magnetic separation unit (CliniMACS, Miltenyi Biotec) with either the Depletion 2.1 (D2.1, n=11) or the novel Depletion 3.1 (D3.1, n=12) program. If indicated, additional CD34+ selections were performed (n=6). Eleven patients received T-cell-depleted grafts after reduced-intensity conditioning.The median log T-cell depletion was better with the D2.1 compared to the D3.1 (log 3.6 vs. log 2.3, p0.05) and was further improved by introducing an immunoglobulin G (IgG)-blocking step (log 4.5 and log 3.4, respectively). The D3.1 was superior to the D2.1 (p0.05) in median recovery of CD34+ cells (90% vs. 78%) and in median recovery of CD3- cells (87% vs. 76%). The median processing times per 10(10) total cells were 0.90 hours (D2.1) and 0.35 hours (D3.1). The transplanted grafts (directly T-cell-depleted products with or without positively selected CD34+ cells) contained a median of 10.5 x 10(6) per kg CD34+, 0.93x10(5) per kg CD3+, and 11.6x10(6) per kg CD56+. Rapid engraftment was achieved in 10 patients. The incidences of acute graft-versus-host disease were less than 10 percent (Grade I/II) and 0 percent (Grade III/IV).The novel D3.1 program with IgG blocking enables highly effective, time-saving large-scale T-cell depletion. Combining direct depletion techniques with standard CD34+ selection enables the composition of grafts optimized to the specific requirements of the patients.

The role of the DEK protein, involved in the leukemia-associated fusion protein DEK-CAN, is not yet known. In this study, we show a higher expression of DEK mRNA in immature cells than in mature cells. Furthermore, a correlation between DEK expression and cell proliferation was demonstrated, suggesting that DEK plays a role in the proliferation of hematopoietic cells and raising the question of whether the DEK-CAN fusion protein might perturb regulation of proliferation in leukemic cells.

Human leukocyte elastase, proteinase 3 and cathepsin G are neutrophil granule proteins belonging to the hematopoietic serine protease superfamily. In addition to their established roles in inflammation, they have recently been implicated as regulators of granulopoiesis and mediators of apoptosis. We set out to characterize the individual biosynthetic profiles of these proteins in a neutrophil differentiation model.CD34+CD38+ hematopoietic progenitor cells from 21 healthy human bone marrow donors were cultured in vitro in the presence of recombinant human granulocyte colony-stimulating factor (G-CSF). Biosynthetic radiolabeling was performed in cells from 13 subjects after various periods of differentiation induction. Following protein extraction, the proteins were specifically immunoprecipitated from cell lysates and media and run in gel electrophoresis. Biosynthetic profiles of azurophil granule proteins, in particular members of the neutrophil serine protease family, were examined during myeloid differentiation.The onset of synthesis of myeloperoxidase, lysozyme, leukocyte elastase, and proteinase 3 occurred early after differentiation induction with G-CSF, while synthesis of cathepsin G, azurocidin, and bactericidal/permeability-increasing protein was detected somewhat later. Cathepsin G and proteinase 3 were retained intracellularly relatively efficiently, while leukocyte elastase and lysozyme were secreted to a greater extent. Cell morphology and positive immunocytochemistry for lactoferrin as well as flow cytometric analysis of selected surface antigens confirmed neutrophil-like maturation.We demonstrate that azurophil granule proteins, including proforms of human leukocyte elastase, proteinase 3 and cathepsin G, are constitutively secreted to various degrees during in vitro myeloid differentiation of human hematopoietic progenitor cells, in addition to being stored intracellularly in active forms. These findings suggest protein-specific sorting mechanisms and may have implications for the regulation of granulopoiesis.

Cathepsin G is a hematopoietic serine protease stored in the azurophil granules of neutrophil granulocytes. The mRNA of cathepsin G is transiently expressed during the promyelocyte stage of neutrophil maturation. The protease plays several roles in inflammatory actions of neutrophils, such as bactericidal effects. A human cathepsin G gene fragment of 6 kb directs a promyelocyte-specific expression in transgenic mice, indicating the presence of necessary cis-acting elements. However, neither the precise architecture of the promoter, nor the trans-acting factors responsible for its activation, have been characterized. In the present work, 2.6 kb upstream of the translation start site of the human cathepsin G gene was cloned. When transfected to monoblast-like U937 or to acute promyelocytic leukemia NB4 cells, both expressing endogenous cathepsin G, the initial 360 bp upstream of the translation start were sufficient to direct a strong expression of a luciferase reporter gene. No expression was observed in erythroid K562 control cells. Further deletions revealed three major regulatory regions containing the consensus binding-sites for the transcription factors C/EBP, c-myb and PU.1. Moreover, a GC-rich region, similar to a cis-element in the proteinase 3 promoter, was identified. Direct binding of the trans-factors C/EBPalpha, C/EBPepsilon, c-myb and PU.1 to the promoter was shown by chromatin immunoprecipitation. The functional significance of the cis-elements was verified by site-directed mutagenesis. Mutations of the putative PU.1 site moderately decreased the activity of the promoter in monoblastic U937 cells, but not in promyelocytic NB4 cells. Separate mutations of the putative C/EBP binding site, c-myb-binding site or the GC-rich element resulted in a dramatically reduced transcriptional activity in both cell lines, suggesting cooperation between corresponding trans-factors.

Neutrophils contain an assembly of granules destined for regulated secretion, each granule type with distinct constituents formed before terminal differentiation. The earliest granules are designated azurophil (primary), followed in time by specific (secondary), and gelatinase granules as well as secretory vesicles. Transcription factors regulate the genes for the granule proteins to ensure that expression of the gene products to be stored in different organelles is separated in time. Similar to lysosomal enzymes, many granule proteins, in particular those of the heterogeneous azurophil granules, are trimmed by proteolytic processing into mature proteins. Rodent myeloid cell lines have been utilized for research on the processing and targeting of human granule proteins after transfection of cDNA. Results from extensive work on the hematopoietic serine proteases of azurophil granules, employing in vitro mutagenesis, indicate that both an immature and a mature conformation are compatible with targeting for storage in granules. On the other hand, the amino-terminal propeptide of myeloperoxidase facilitates both the export from the endoplasmic reticulum and targeting for storage in granules. Similarly, targeting of defensins rely on an intact propeptide. The proteolytic processing into mature granule protein is most commonly a post-sorting event. Mis-sorting of specific granule proteins into azurophil or lysosome-like granules can result in premature activation and degradation, but represents a potential for manipulating the composition and function of neutrophil granules.

The neutrophil granulocytes have a critical role in innate immunity in particular through phagocytosis of microorganisms. Their cytoplasmic granule subsets store digestive enzymes and antibiotic proteins that are released during this event. During the passage in the secretory pathway the granule proteins are retrieved from constitutive secretion to become sorted for storage. The aim of this thesis was to understand retrieval and sorting for storage through studying processing and sorting of MPO, BPI and hCAP-18 and exogenous secretory proteins. We transfected cDNA of these human proteins to the murine myeloid RBL and 32D cell lines. Mutations of cDNA were made before transfection. A propeptide- deleted MPO precursor was not processed to mature MPO but degraded or secreted, indicating that the propeptide is a prerequisite for final processing and targeting of proMPO to granules. Chimeras between the MPO propeptide and secretory proteins prolonged the ER retention compared with that of the native proteins. Thus, the propeptide may mediate the long ER-residence of proMPO. The amino-terminal half of BPI, when expressed alone, was targeted for storage, while the carboxy-terminal half was not, but, it increased the stability of BPI and chimeric proteins, indicating that this half is important for stability while the amino-terminal half may be important for sorting. The non-myeloid LBP and alpha1-microglobulin were targeted to, but unstable, in granules, indicating cell-specific rather than protein-specific sorting in the cells. But not all proteins were sorted, e g sTNFR1 and propeptide-deleted MPO were secreted, suggesting that physico-chemical properties affect sorting. hCAP-18 was targeted, i e missorted, to the lysosome-related organelles of RBL cells with subsequent prematurely processing and degradation. Thus, the results of the thesis indicate cell-specific retention mechanisms for sorting for storage in myeloid cells.

Siblings who are asked to donate haematopoietic stem cells are in a vulnerable position. Potential sibling donors must be fully informed, orally and in writing, and be able to provide informed consent without pressure or coercion.1, 2, 3 To achieve this, one must focus on the management of confidentiality in terms of donor volunteer status and strive to protect the integrity of the sibling.4, 5 At our centre we decided to improve this aspect in the care of potential sibling donors, enabling an early termination of the process for those not willing or unable to donate. We also wanted to improve the information to, and the follow-up of sibling donors.

Neutrophil granulocytes are specialized phagocytic cells that carry a collection of granules for regulated secretion, each with distinct constituents. The granules can be classified as azurophil (primary), developed first, followed in time by specific (secondary) granules gelatinase granules, and secretory vesicles. Stage- and tissue-specific transcription factors govern the successive expression of genes for granule proteins to allow storage of the gene products in these organelle categories whose packaging is separated in time. Many of the granule proteins, in particular those of the heterogeneous lysosome-like azurophil granules, are subject to extensive post-translational proteolytic processing into mature proteins, most commonly as a post-sorting event. A selective aggregation of proteins destined for storage in granules, as discussed in this review, would facilitate their retention and eliminate a need for distinct sorting motifs on each granule protein. Aggregation of granule proteins, that are often cationic, would be assisted by the anionic serglycin proteoglycans present in neutrophils. The antibacterial granule proteins can serve as models for antibiotics and some of them possess a potentially useful therapeutic ability to bind and neutralize endotoxin. Because aberrant expression of transcription factors regulating the synthesis of granule proteins is often found in leukemia, the clarification of mechanisms regulating the timed expression of granule proteins will shed light on the maturation block in myeloid leukemias.

Proteinase 3 (PR3) is a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors. The protease has been identified as an autoantigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) occurring in patients with Wegener's granulomatosis. To characterize the biosynthesis and processing of human PR3 in a transgenic cellular model, cDNA encoding human pre-proproteinase 3 cloned from U-937 cells was transfected to the rat basophilic/mast cell line RBL-1 and the murine myeloblast-like cell line 32D cl3. The stable expression of transgenic proteinase 3 was characterized by biosynthetic labeling, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. After pulse labeling for 30 min two proforms of PR3 (32 and 35 kDa), differing in carbohydrate content but with protein cores of identical size, were demonstrated. Chase of the label resulted in a processed 32-kDa form clearly visible in RBL, but only faintly in 32D cells, probably indicating delayed intracellular transfer in the latter cell line. Partial digestion with N-glycosidase F showed that both potential N-glycosylation sites on PR3 were occupied and conversion of the oligosaccharide side chains into complex forms was demonstrated by acquisition of resistance to endoglycosidase H. Translocation of PR3 to granules was shown by subcellular fractionation and immunocytochemistry. Enzymatic activation of PR3 was suggested by affinity to diisopropylfluorophosphate and removal of an amino-terminal propeptide. Cells transfected with PR3 showed positive immunofluorescence for ANCA-containing sera from patients with Wegener's granulomatosis. Our results show that human PR3 transfected to RBL or 32D cells is synthesized as a 29-kDa protein core glycosylated on two distinct sites. Oligosaccharide trimming and proteolytic processing occur and the protein is targeted for granular storage in a form antigenic for ANCA.

The hematopoietic neutral serine proteases leukocyte elastase and cathepsin G are synthesized as inactive precursors, but become activated by removal of an amino-terminal dipeptide and are stored in granules. Moreover, the pro forms of elastase and cathepsin G show carboxyl-terminal prodomains of 20 and 11 amino acids, respectively, which are not present in the mature enzymes. To investigate mechanisms for processing, activation, and granular targeting, we have utilized transgenic expression of myeloid serine proteases in the rat basophilic/mast cell line RBL-1 (Gullberg, U., Lindmark, A., Nilsson, E., Persson, A.-M., and Olsson, I.(1994) J. Biol. Chem. 269, 25219-25225). Leukocyte elastase was stably expressed in RBL-1 cells, and the translation products were characterized by biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Processing of a main pro form of 34 kDa into mature 31- and 29-kDa forms was demonstrated. Translocation of mature forms to granule-containing fractions was shown by subcellular fractionation experiments. The processed forms were enzymatically active, judging by the occurrence of amino-terminal processing demonstrated by radiosequence analysis, the acquisition of affinity for the protease inhibitor aprotinin, and the appearance of elastase activity in transfected RBL cells. To investigate the function of the carboxyl-terminal prodomains, deletion mutants of leukocyte elastase and cathepsin G lacking the carboxyl-terminal extension were constructed and transfected into RBL cells. Our results show that as full-length proteins, the deletion mutants were converted to active enzymes and transferred to granules with kinetics similar to that of wild-type enzymes. We conclude that human leukocyte elastase and cathepsin G are converted into enzymatically active forms when expressed in RBL cells and targeted for storage in granules; the carboxyl-terminal prodomains are necessary neither for enzymatic activation nor for targeting to granules in RBL cells.

The azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.