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Fifty two serum samples from patients with Crohn's disease, 24 from patients with ulcerative colitis, and 12 from patients with primary sclerosing cholangitis were analysed for the presence of anti-neutrophil cytoplasm antibodies (ANCA) of IgG and IgA class by means of enzyme linked immunosorbent assays using lactoferrin, myeloperoxidase, and antigen extracted from azurophil granules, 'alpha antigen' (that is, an antigen preparation containing proteinase 3) as substrates. A high frequency of positive tests for IgG anti-lactoferrin antibodies was found in sera from patients with ulcerative colitis (50%) and primary sclerosing cholangitis (50%). In Crohn's disease only 4 of 52 (8%) sera had anti-lactoferrin antibodies--in all four instances the sera belonged to patients with disease involving the colon. All patients with sclerosing cholangitis and positive tests for anti-lactoferrin had ulcerative colitis. Low levels of IgG antibodies against myeloperoxidase or alpha antigen were also found occasionally in sera from patients with ulcerative colitis and primary sclerosing cholangitis. IgA antibodies directed against lactoferrin and alpha antigen (but not myeloperoxidase) were seen in all three conditions. [ABSTRACT FROM PUBLISHER]

A truncated form of annexin I, formed during Ca2+-induced translocation to neutrophil specific granules and secretory vesicles/plasma membranes, is generated through the action of an endogenous membrane protease. The cleavage of annexin I is inhibited by the metalloprotease inhibitor 1,10-phenanthroline as well as by Triton X-100 and dithiothreitol, classifying the protease as a membrane-bound, thiol-dependent metalloprotease. The cleavage site is located close to the N-terminal of annexin I, leaving a truncated form of the molecule, des1-8 annexin I, that contains the Ca2+-binding sites, as well as a number of phosphorylation sites of importance for the function of the protein. When assessing binding capacity to different neutrophil organelles, full-length annexin I bound to azurophil granules, specific granules, and secretory vesicles/plasma membranes, while des1-8 annexin I only bound to specific granules and secretory vesicles/plasma membranes, but not to azurophil granules (C. Sjölin, C. Dahlgren, Biochim. Biophys. Acta 1281 (1996) 227-234). This implies that there are different mechanisms of binding to neutrophil organelles of full-length annexin I and the truncated form, and that cleavage of annexin I might be of regulatory importance for the degranulation process.

Neutrophil production of reactive oxygen species is dependent on an assembly process that involves a translocation of the cytosolic NADPH-oxidase components (p47phox; p67phox; Rac2) to a b cytochrome containing membrane. Based on the fact that an intracellular Ca2+ rise can activate the oxidase without any extracellular release of reactive oxygen species, we suggest that the oxidase can be assembled in a membrane distinct from the plasma membrane. Disintegrated cells were used to monitor Ca2+ dependent membrane binding of neutrophil cytosolic proteins. Membranes containing the b cytochrome part of the oxidase, i.e., specific granules and plasma membranes/secretory vesicles, were used in the translocation experiments. Several cytosolic proteins were found to translocate to specific granules as well as the plasma membranes/secretory vesicles, one of them being annexin I. Using antibodies in the blotting assay against the cytosolic oxidase components p47phox and p67phox, we could show that no Ca2+ dependent translocation of these cytosolic proteins occur to neither of the b cytochrome containing membranes.

In an earlier study, annexin XI was found to be present in the cytosol of neutrophil granulocytes (Blood (1996) 87, 4817). The protein was isolated by calcium-dependent translocation to specific granules and was found to be a 42-kDa truncated form of annexin XI. Using human autoantibodies directed against annexin XI we have now reinvestigated the ability of full size annexin XI to translocate to different neutrophil organelles isolated by subcellular fractionation. The autoantisera used recognised a protein of 55-kDa in neutrophil cytosol and comparison with a whole cell lysate indicated that the larger portion of the cellular content of this protein is localised to the cytosol. Azurophil granules, specific granules and secretory vesicles/plasma membrane were isolated by subcellular fractionation on Percoll gradients, mixed respectively with neutrophil cytosol and the calcium concentration was raised. Immunoblotting showed that annexin XI translocated to specific granules and secretory vesicles/plasma membrane at 100 micromol/l calcium. When raising the concentration of calcium to 1 mmol/l, annexin XI translocated to the azurophil granules as well. Periphagosomal translocation of annexin XI occurred during phagocytosis of yeast particles, implying that this protein plays a role in the events associated with the phagocytic process.

A neutrophil annexin I-related protein, detected after translocation of cytosolic proteins to specific granules and secretory vesicles/plasma membrane (Sjölin et al. (1994) Biochem. J. 300, 325-330), has been characterized with respect to origin and organelle-binding properties. The annexin I-related protein is formed as a result of annexin I cleavage, and this occurs during translocation of annexin I to the specific granules and secretory vesicles/plasma membrane, but not when annexin I is translocated to azurophil granules. The cleavage required calcium and it was facilitated in the presence of specific granules or secretory vesicles/plasma membrane, but not in the presence of azurophil granules. We conclude that the membranes of specific granules and secretory vesicles/plasma membrane contain a protease which is able to cleave annexin I into a truncated 38 kDa fragment, which retains the ability to bind to these organelles. The azurophil granules lack the capacity to cleave annexin I as well as the ability to bind the 38 kDa fragment. These findings may implicate a role for annexin I in the divergent regulation of exocytosis of the different neutrophil granules.

Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granules. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti-cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 micromol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N-terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI.

When neutrophils and macrophages phagocytose a prey, e.g., complement (C3b)-opsonized yeast particles, the oxygen radical generating NADPH-oxidase is activated. In neutrophils, most of the production of oxygen metabolites occurred in an intracellular compartment, possibly in the phagolysosome. In contrast, no intracellular production could be detected in human macrophages. In these cells, the subcellular localization of the superoxide-generating NADPH-oxidase and associated cytochrome b was assessed in intact cells with indirect immunofluorescence and confocal laser scanning microscopy, and with subcellular fractionation, using centrifugation on Percoll density gradients. A dual localization of the cytochrome b as well as the dormant NADPH-oxidase activity in neutrophils was in agreement with earlier immunocytochemical, biochemical, and subcellular fractionation studies. Furthermore, most of the activity was recovered from the specific granules, whereas only a small fraction was retained in the plasma membrane. In contrast, the cytochrome b/NADPH-oxidase activity in macrophages localized primarily in the plasma membrane fraction. We suggest that the macrophages are incapable of producing reactive oxygen species intraphagosomally, due to an absence of a granule-localized pool of the membrane components of the NADPH-oxidase.

Using slot blot, we show that myeloperoxidase (MPO), a constituent of azurophil granules of neutrophil polymorphonuclear leukocytes, can be measured quantitatively using a commercially available chemiluminescence kit, originally developed for detection of specific proteins on Western blots. MPO is determined through its ability to catalyze the oxidation of luminol, resulting in the emission of light which is recorded on a photographic film. The sensitivity of the method is high and allows MPO from less than 100 cells to be detected. This method was used to determine MPO in exudate fluid and in neutrophil fractions following disintegration and subcellular fractionation of the postnuclear supernatant on two-layer Percoll gradients.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of circulating anti-neutrophil cytoplasm antibodies (ANCA), which are defined by a diffuse, granular staining of the cytoplasm of alcohol-fixed human neutrophils by indirect immunofluorescence (IIF). Detection of antineutrophil cytoplasm antibodies has a high sensitivity and specificity for active Wegener's granulomatosis (WG) and reflects the effect of treatment. In the present enzyme-linked assay, immunoplates were coated with the cytoplasmic alpha fraction of neutrophils obtained from apparently healthy human donors by nitrogen bomb cavitation and subsequent Percoll gradient centrifugation. Alkaline phosphatase-labelled anti-human IgG was used as a secondary antibody. Diluted sera from 70 patients with WG and 16 patients with other diseases with anti-myeloperoxidase antibodies (anti-MPO) were examined. It is concluded that the ELISA accurately detects IIF ANCA positive patients with WG, is helpful in detecting WG patients in remission, is not influenced by the presence of anti-MPO and may help in detecting ANCA in cases with granulocyte-specific anti-nuclear antibodies since this IIF pattern obscures the IIF ANCA patterns. The ELISA with titration can be carried out in 3.5 h whereas a rapid test just to detect ANCA can be performed in 30 min.

Sixty-eight patients with hip fracture, mean age 79 years, from the city of Lund, were studied with special reference to functional and social rehabilitation at follow-up 1 year later. As compared with patients coming from institutions for permanent care, patients coming from their own homes had a significantly better prognosis in terms of survival, mobility and ability to cope with activities of daily living (ADL). However, patients returning home needed increased domestic help. Patients living with someone returned home sooner than those living alone. Although many of the patients who returned home could walk without support or with a walking-stick, more than one half did not go out shopping. More active measures, e.g. early home visits by a rehabilitation team, might give the patients more self-confidence and independence. Immediate weight-bearing did not appear to impair healing of the hip fracture or increase the risk of necrosis of the femoral head.