Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-selectin glycoprotein ligand-1 by neutrophil elastase and cathepsin G

In the inflammatory response, leukocyte rolling before adhesion and transmigration through the blood vessel wall is mediated by specific cell surface adhesion receptors. Neutrophil rolling involves the interaction of P-selectin expressed on activated endothelium and its counter-receptor on neutrophils, P-selectin glycoprotein ligand-1 (PSGL-1). Here, it is reported that P-selectin binding to neutrophils is lost under conditions that cause the release of proteinases from neutrophil primary granules. Treatment of neutrophils with the purified neutrophil granule proteinases, cathepsin G and elastase, rapidly abolished their capacity to bind P-selectin. This inactivation corresponded to loss of the N-terminal domain of PSGL-1, as assessed by Western blot analysis. A loss of intact PSGL-1 protein from the surfaces of neutrophils after the induction of degranulation was also detected by Western blot analysis. Cathepsin G initially cleaved near the PSGL-1 N-terminus, whereas neutrophil elastase predominantly cleaved at a more C-terminal site within the protein mucin core. Consistent with this, cathepsin G cleaved a synthetic peptide based on the PSGL-1 N-terminus between Tyr-7/Leu-8. Under conditions producing neutrophil degranulation in incubations containing mixtures of platelets and neutrophils, the loss of PSGL-1, but not P-selectin, from platelet-neutrophil lysates was detected. Cathepsin G- or neutrophil elastase-mediated PSGL-1 proteolysis may constitute a potential autocrine mechanism for down-regulation of neutrophil adhesion to P-selectin.     

The secretion of insulin-like growth factor I, prolactin and insulin-like growth factor binding protein 1 by the decidua as predictors of human fetal growth.

To determine if in vitro secretion of the decidual peptides insulin-like growth factor I (IGF-I), prolactin or insulin-like growth factor binding protein 1 (IGFBP-1) correlates with infant birthweight in uncomplicated, term human pregnancies, decidua from 45 pregnancies with normally distributed birthweights was cultured in defined medium for 24 h. IGF-I, prolactin and IGFBP-1 concentrations in the culture medium were measured by radioimmunoassay. Neither infant birthweight nor a normalized measure of infant birthweight (birthweight z-score) correlated with the quantity of IGF-I, prolactin or IGFBP-1 secreted by the decidua from that pregnancy. There were no differences in any of the peptide hormones assayed when the pregnancies were grouped by infant sex. IGF-I and prolactin secretion by individual decidual samples correlated positively. IGF-I and IGFBP-1 secretion also correlated positively in individual samples. A previously identified correlation between decidual IGF-I secretion and infant birthweight among a group of normal and growth restricted (IUGR) pregnancies was not confirmed in the current study. These data indicate that the decrease in decidual IGF-I and prolactin secretion seen in IUGR pregnancies is not the hormone profile of the low birthweight end of a normal population, but a distinct endocrine profile.     

Physical activity, insulin-like growth factor 1, insulin-like growth factor binding protein 3, and survival from colorectal cancer

BACKGROUND: Recent reports have shown that physical activity improves the outcome of patients with colorectal cancer as well as breast and prostate cancer. However, the mechanisms whereby physical activity reduces cancer mortality are not well established. METHODS: Incident cases of colorectal cancer were identified among participants of the Melbourne Collaborative Cohort Study, a prospective cohort study of 41,528 Australians recruited from 1990 to 1994. Information on tumour site and stage, treatments given, recurrences, and deaths were obtained from systematic review of the medical records. Baseline assessments of physical activity and body size were made, and cases with available plasma had pre-diagnosis insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) levels measured. We assessed associations between these hormones and colorectal cancer specific deaths with respect to physical activity. RESULTS: A total of 526 cases of colorectal cancer were identified, of which 443 had IGF-1/IGFBP-3 levels measured. Median follow up among survivors was 5.6 years. For the physically active, increasing IGFBP-3 by 26.2 nmol/l was associated with a 48% reduction in colorectal cancer specific deaths (adjusted hazard ratio (HR) 0.52 (0.33-0.83); p = 0.006). No association was seen for IGF-1 (adjusted HR 0.90 (0.55-1.45); p = 0.65). For the physically inactive, neither IGF-1 nor IGFBP-3 was associated with disease specific survival. CONCLUSIONS: This study supports the hypothesis that the beneficial effects of physical activity in reducing colorectal cancer mortality may occur through interactions with the insulin-like growth factor axis and in particular IGFBP-3.     

Insights from insulin-like growth factor binding protein transgenic mice

The existence of abundant high affinity binding proteins for the IGFs, the IGF binding proteins (IGFBPs), was first demonstrated more than 40 yr ago in the very early days of somatomedin research. With the development of molecular techniques and transgenic and knockout mouse models, the nature, complexity, and redundancy of the IGFBPs have now started to be elucidated. Indeed the functional role of the circulating IGFs and the originally proposed endocrine somatomedin hypothesis have recently been questioned. The limited reports to date indicate that IGFBP knockout mice have few phenotypic manifestations. In contrast, overexpression of IGFBPs in transgenic mice is associated with manifestations that provide some insight into the physiological role of the binding proteins. The predominant effect of generalized or tissue-specific overexpression of the IGFBPs has been growth inhibition as would be anticipated from inhibition of the actions of IGF-I and -II. In addition, impaired glucose homeostasis and reduced fecundity have been observed in both IGFBP-1- and IGFBP-3-overexpressing transgenic mice. This review examines the data reported to date for transgenic mouse models that overexpress IGFBPs. In addition, data from transgenic mice that overexpress the acid-labile subunit, an important component of the ternary complex, have also been reviewed.     

IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G

Interleukin-33 (IL-33) (NF-HEV) is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to important diseases, including asthma, rheumatoid arthritis, ulcerative colitis, and cardiovascular diseases. IL-33 signals through the ST2 receptor and drives cytokine production in type 2 innate lymphoid cells (ILCs) (natural helper cells, nuocytes), T-helper (Th)2 lymphocytes, mast cells, basophils, eosinophils, invariant natural killer T (iNKT), and natural killer (NK) cells. We and others recently reported that, unlike IL-1β and IL-18, full-length IL-33 is biologically active independently of caspase-1 cleavage and that processing by caspases results in IL-33 inactivation. We suggested that IL-33, which is released upon cellular damage, may function as an endogenous danger signal or alarmin, similar to IL-1α or high-mobility group box 1 protein (HMGB1). Here, we investigated the possibility that IL-33 activity may be regulated by proteases released during inflammation. Using a combination of in vitro and in vivo approaches, we demonstrate that neutrophil serine proteases cathepsin G and elastase can cleave full-length human IL-33(1-270) and generate mature forms IL-33(95-270), IL-33(99-270), and IL-33(109-270). These forms are produced by activated human neutrophils ex vivo, are biologically active in vivo, and have a ~10-fold higher activity than full-length IL-33 in cellular assays. Murine IL-33 is also cleaved by neutrophil cathepsin G and elastase, and both full-length and cleaved endogenous IL-33 could be detected in the bronchoalveolar lavage fluid in an in vivo model of acute lung injury associated with neutrophil infiltration. We propose that the inflammatory microenvironment may exacerbate disease-associated functions of IL-33 through the generation of highly active mature forms.     

Serum growth hormone binding protein (GHBP) and insulin-like growth factor binding protein (IGFBP)]

Insulin--like growth factors bind to specific binding proteins (IGFBPs) in serum and tissues. At present, six different IGFBPs have been characterized. Recent studies suggest that IGFBPs act as a reservoir for IGFs but also modulate the bioavailability of IGFs. Binding protein for growth hormone (GH) in serum has been recognized recently. Interestingly, the high affinity GH binding protein (GHBP) is identical with the extracellular domain of GH receptor and is absent in patients with Laron-type dwarfism, suggesting that serum GHBP might serve as a marker for the GH receptor in tissue. In this short review, updated information on serum GHBP and IGFBP is presented.     

Total and free insulin-like growth factor I, insulin-like growth factor binding protein 3 and acid-labile subunit reflect clinical activity in acromegaly

The aim was to evaluate, markers of disease activity in acromegaly in relation to perceived disease activity. Thirty-seven consecutively treated, acromegalic patients, classified by clinical symptoms as inactive (n=16), slightly active (n=10) and active (n=11), entered the study. When evaluating the inactive and the active groups, we found that positive and negative predictive values (PV(pos), PV(neg)) for clinical disease activity of total and free insulin-like growth factor-I (IGF-I) were 0.59, 0.90 and 1.00, 0.82 respectively. Acid-labile subunit (ALS) showed diagnostic merit similar to insulin-like growth factor binding protein-3 (IGFBP-3) with PV(pos) of 0.69 and 0.71 and PV(neg) of 0.91 and 0.92 respectively. We conclude that free IGF-I is more closely related than total IGF-I to perceived disease activity and is as such useful when evaluating previously treated acromegaly for disease activity. Total IGF-I, IGFBP-3 and ALS possess a higher PV(neg) for the clinical disease activity. None of the parameters can at present be claimed to be superior to the others and thus all the measured parameters are recommended to be part of the evaluation of acromegalic patients.     

The insulin-like growth factor binding proteins in uncultured human cartilage: increases in insulin-like growth factor binding protein 3 during osteoarthritis

OBJECTIVE: To assess changes in the insulin-like growth factor binding proteins (IGFBPs) in uncultured cartilage during stages of osteoarthritis (OA), and to determine if OA cartilage is capable of autocrine secretion of IGFBPs. METHODS: Articular cartilage was dissected from fibrillated and nonfibrillated sites of 11 human femoral heads, and extracted in buffer containing 8M urea. IGFBPs were identified by immunoprecipitation and subsequent analysis by (125)I-IGF-2 Western ligand blotting (WLB), radioimmunoassay, or 2-site immunoradiometric assay (IRMA). IGFBPs were assessed in cartilage extracts by WLB. IGFBP-3 content was determined by IRMA and synthesis by metabolic labeling with (35)S-cysteine in organ cultures. RESULTS: Sample grouping into 3 distinct OA strata was supported by gross pathology of the femoral heads, histologic grading of cartilage slices, and biochemical analysis of the glycosaminoglycan and protein content of the extracts. Group I was normal/mild OA, group II was intermediate OA, and group III was severe OA. IGFBP-2 was present in all samples, IGFBP-4 in sporadic samples, and BP-3 in group II-III samples. By IRMA, group I had a mean +/- SD of 6.26 +/- 2.6 ng IGFBP-3/mg soluble protein (IGFBP-3) (n = 6), group II had a mean +/- SD 14 +/- 7.5 IGFBP-3 (n = 10), and group III had a mean +/- SD 17.03 +/- 8.94 IGFBP-3 (n = 6). Analysis of variance showed group differences (F[3,19] = 3.84, P = 0.04), and post hoc tests revealed that IGFBP-3 levels were higher for group III versus group I (P = 0.04). OA cartilage synthesized IGFBP-3. CONCLUSION: Increases in net cartilage content of IGFBP-3 occurred in intact OA cartilage, reaching statistically significant elevation in severe disease. There was autocrine IGFBP-3 production in OA cartilage.     

Heterogeneity of binding subunits of the human 150K insulin-like growth factor binding protein.

Models for the structure of the GH-dependent 150K insulin-like growth factor-binding protein (IGF-BP) complex include 1) a binding subunit of 40-60K mol wt associated with a larger nonbinding component, and 2) an oligomeric structure simply made up of six 25-28K monomeric IGF-BP complexes. To evaluate these alternative models we examined the IGF-binding characteristics and behavior on an SP-Sephadex ion exchange column of BP species identified by chemically cross-linking [125I]IGF-I and [125I]IGF-II. In addition, human serum was gel filtered on Sephadex G-200 in 0.05 M NH4HCO3, pH 8.0, and the 150K BP identified by binding of [125I]IGF-II to column fractions. When [125I]IGF-I or [125I]IGF-II was cross-linked to the 150K BP with disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10-15%) and autoradiography, four specifically labeled complexes of 20K, 24K, 33K, and 47K mol wt were identified. We examined the IGF-binding characteristics of these species by cross-linking [125I]IGF-I and [125I]IGF-II after incubation in the presence of increasing concentrations of unlabeled IGF-I or IGF-II. Formation of the 24K complex was inhibited more potently by IGF-II than IGF-I, whereas the relative potency of IGF-I vs. IGF-II for inhibition of the formation of the other complexes depended upon whether [125I]IGF-II or [125I]IGF-I was used. When the 150K BP complex generated from gel filtration on Sephadex G-200 was acid stripped, the only species seen with chemical cross-linking of either [125I]IGF-I or [125I]IGF-II was the 47K complex. By both conventional competitive binding studies and cross-linking [125I]IGF-I and [125I]IGF-II after incubation with increasing concentrations of unlabeled IGF-I or IGF-II, the formation of the 47K complex was usually more potently inhibited by IGF-I than IGF-II. When Cohn fraction IV extract was chromatographed on a SP-Sephadex column (pH 3) and cross-linking performed on the flow-through, the 47K species was intensely labeled, and the 20K, 33K, and 24K complexes were weakly labeled or not seen at all. The 20K, 33K, and 24K complexes could be identified after cross-linking [125I]IGF-II to the pH 7 eluate from the ion exchange column, but the 47K complex was not seen. These data provide additional physical evidence for separate binding sites with different relative affinities for IGF-I and IGF-II within the 150K BP complex.     

Serum insulin-like growth factor-I, insulin-like growth factor binding proteins, and bone mineral content in hyperthyroidism.

The mechanism by which thyroid hormones promote bone growth has not yet been elucidated. In vitro, thyroid hormones stimulate insulin-like growth factor-I (IGF-I) production by osteoblasts, which is important for the anabolic effects of the hormone on bone. To determine whether the IGF-I/IGF binding protein (IGFBP) profile is affected when thyroid hormone production is altered in vivo, we studied 36 women who had recently been diagnosed with hyperthyroidism (age: 29-67 years; 19 with Graves' disease, 17 with toxic nodular goiter) and 36 age-matched healthy women as controls. Serum IGF-I, and its binding proteins (IGFBP-3, IGFBP-4, and IGFBP-5), as well as bone mineral density (BMD) at the lumbar spine, femoral neck, and radius midshaft were measured before and 1 year after antithyroid (methimazole) treatment. Serum IGF-I levels were significantly increased in the hyperthyroid patients before treatment (214 +/- 18.2 ng/mL vs. 145 +/- 21.3 ng/mL; p < 0.05). There was no difference in IGF-I levels of patients with Graves' disease and toxic nodular goiter. Serum IGF-I concentrations returned to normal after treatment with methimazole. Serum IGFBP-3 and IGFBP-4 values were significantly elevated in the hyperthyroid group before treatment (3960 +/- 220 ng/mL and 749.7 +/- 53.1 ng/mL vs. 2701 +/- 180 ng/mL and 489.9 +/- 32.4 ng/mL; p < 0.05 and p < 0.01, respectively) and were reduced to those of controls after treatment. Serum IGFBP-5 of hyperthyroid subjects was not different from that of controls either before or after therapy. Serum free thyroxine showed a positive correlation with serum levels of IGF-I (r = 0.73, p < 0.05), IGFBP-3 (r = 0.59, p < 0.05), and IGFBP-4 (r = 0.67, p < 0.05) but not IGFBP-5. BMD at the radius midshaft was significantly lower in hyperthyroid patients at the start of the study and showed a positive correlation with serum IGF-I (r = 0.58; p < 0.001) and a negative correlation with IGFBP-4 (r = -0.61; p < 0.05). Radius BMD showed a 7.2% increase in the hyperthyroid group after 1 year of methimazole treatment, and the correlation between BMD and serum IGF-I disappeared. Our data indicate that thyroid hormones may influence the IGF-I/IGFBP system in vivo in hyperthyroidism. The anabolic effects of increased levels of IGF-I may be limited in hyperthyroidism due to the increases of inhibitory IGFBPs that can counteract the anabolic effects and contribute to the observed net bone loss.     

Effects of luteinizing hormone, insulin, insulin-like growth factor-I and insulin-like growth factor small binding protein 1 in the polycystic ovary syndrome.

This study explores the clinical and endocrine implications of hyperinsulinaemia in the polycystic ovary syndrome (PCOS). Oral glucose tolerance tests were performed on 34 lean and 19 obese women with PCOS and on 13 lean women with normal ovaries. Insulin measurements were compared with basal gonadotrophins, androgens, insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein 1 (IGFBP-1). Unselected lean women with PCOS were found to have fasting hyperinsulinaemia and the raised serum insulin concentrations were associated with menstrual disturbance and hyperandrogenaemia. In addition, serum insulin concentrations in lean women with PCOS correlated positively with serum IGF-I and negatively with serum IGFBP-1 concentrations. Ovarian stimulation by insulin appears to be independent of luteinizing hormone (LH) and is an important feature in 30% of lean women with PCOS.     

Follicular fluid insulin-like growth factor binding protein profiles in polycystic ovary syndrome.

Insulin-like growth factor binding proteins (IGFBPs) are believed to modulate the actions of IGF-I and IGF-II at the cellular level. We have examined, by Western ligand blot analysis, the IGFBP profiles in follicular fluid (FF) from patients with polycystic ovarian syndrome (a disorder of ovarian folliculogenesis), compared to FF from atretic and developing (estrogenic) follicles from normally cycling women. IGFBPs with apparent mol wts (Mr) of 41.5, 38.5, 31, 28, and 24kDa were detected in PCOS FF. The profile of IGFBPs in PCOS FF was indistinguishable from that seen in atretic follicles in cycling women. However, higher levels of the 31, 28, and 24kDa IGFBPs were observed in PCOS FF, compared to healthy, estrogenic follicles. Using specific antisera, the 41.5 and 38.5kDa IGFBPs were identified as IGFBP-3, and the 31kDa IGFBP as IGFBP-2. IGFBP-1, however, was not appreciably detectable in PCOS FF, by Western ligand blotting. Endoglycosidase F treatment of FF decreased the Mr of the 28kDa IGFBP to 24kDa, and neither the 28kDa nor the 24kDa IGFBP was immunoprecipitated by antibodies to IGFBP-1, -2, or -3. Elevated levels of 28kDa and 24kDa IGFBPs in PCOS FF may represent glycosylated and core forms of IGFBP-4. The data presented herein show that in PCOS FF, as well as in FF from atretic follicles from normally cycling women, IGFBP-2 and 28 and 24kDa IGFBPs are present in greater amounts, compared to levels in FF from healthy, developing, estrogenic follicles. One or more of the IGFBP species elevated in atretic and PCOS follicles may bind IGFs in FF, thereby inhibiting IGF action on the granulosa during normal folliculogenesis.     

Identification of vitronectin as a novel insulin-like growth factor-II binding protein.

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(1-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/IGF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.     

Degradation of human factor X by human polymorphonuclear leucocyte cathepsin G and elastase

Cathepsin G and elastase from human polymorphonuclear leucocytes were used in vitro to digest human factor X. Clotting assays showed that both proteinases affected a rapid loss in the coagulant activity of factor X. Calcium ions almost totally protected the coagulant activity of factor X against the action of cathepsin G but not elastase. Polyacrylamide gel electrophoresis (in nonreducing conditions and in the presence of SDS) indicated that the proteolytic action of cathepsin G led to the removal of a peptide of low molecular mass (pX) with the consequent formation of a single stable high molecular mass product (PX). SDS electrophoresis (under reducing conditions and in the presence of SDS) indicated that a pX was derived from the light chain of factor X. The proteolytic action of elastase led to the formation of numerous degradation products. Analysis of the products generated by the action of cathepsin G indicated that cathepsin G cleaved position Phe40:Trp41 in the light chain of factor X. In the presence of citrated plasma, cathepsin G but not elastase, was responsible for a loss in coagulant activity.     

Plasma insulin-like growth factor 1, insulin-like growth factor binding protein 3, and risk of colorectal cancer: a prospective study in northern Sweden

BACKGROUND: Insulin-like growth factor 1 (IGF-1) has antiapoptotic and mitogenic effects on various cell types, and raised IGF-1 levels are increasingly being implicated as potential risk factors for cancer. AIMS: To examine the relationship between IGF-1 and its major plasma binding protein, IGF binding protein 3 (IGFBP-3), and the risk of colorectal cancer. METHODS: We conducted a case-control study nested within the Northern Sweden Health and Disease Cohort. IGF-1 and IGFBP-3 were measured in prediagnostic plasma samples from 168 men and women who developed cancers of the colon (n=110) or rectum (n=58), and from 336 matched controls. RESULTS: Conditional logistic regression analyses showed an increase in colon cancer risk with increasing levels of IGF-1 (odds ratios (ORs) 1.00, 1.89, 2.30, 2.66; p(trend)=0.03) and IGFBP-3 (ORs 1.00, 0.91, 1.80, 1.93; p(trend)=0.02). Rectal cancer risk was inversely related to levels of IGF-1 (ORs 1.00, 0.45, 0.33, 0.33; p(trend)=0.09) and IGFBP-3 (ORs 1.00, 0.75, 0.66, 0.49; p(trend)=0.21). Mutual adjustments between IGF-1 and IGFBP-3 did not materially alter these relationships. CONCLUSIONS: These results support earlier findings of increased risk of colon cancer in subjects with elevated plasma IGF-1. Our results however do not support the hypothesis that the risk of rectal cancer could also be directly related to IGF-1 levels.     

Characterization of urinary insulin-like growth factor binding proteins.

The insulin-like growth factors (IGFs) are important mitogens that are present in many body fluids, where they are commonly bound with high affinity to IGF binding proteins (IGFBPs). We investigated human urine for the presence of IGFBPs. Western ligand blots of concentrated, dialyzed normal urine disclosed the presence of two major bands with IGF binding activity, one at 40-44 kilodaltons and another at 31 kDa. Deglycosylation with endoglycosidase F, and immunoprecipitation with alpha HEC1 antibody revealed these proteins to be hIGFBP-3 and hIGFBP-2, respectively. Comparison of IGFBPs in normal serum and urine showed a reversal of the hIGFBP-2/hIGFBP-3 ratio in urine compared to serum, with hIGFBP-2 being the predominant binding protein in normal urine. The 150 kDa form of hIGFBP-3 was absent in normal urine. In patients with renal disease, the urinary IGFBP (U-IGFBP) pattern was altered. Patients with glomerular disease and proteinuria had elevated U-hIGFBP-3, whereas patients with renal failure who displayed increased urinary beta-2-microglobulin had a dramatic increase in U-hIGFBP-1, in the face of normal serum IGFBP profiles. In conclusion, we have documented the presence of IGFBPs in the urine of normal and diseased individuals. The presence of IGFBPs in urine may complicate the assessment of IGF measurements in urine. U-IGFBPs may be potential clinical markers in renal diseases. Additional studies are required before the origin of urinary IGFBPs in both normal and pathological conditions will be definitively established.     

Human leukocyte elastase, cathepsin G, and lactoferrin: family of neutrophil granule glycoproteins that bind to an alveolar macrophage receptor.

Interactions between polymorphonuclear neutrophils and mononuclear phagocytes are potentially of great importance in a variety of inflammatory processes. As part of a continuing effort to elucidate the physiologic importance of human alveolar macrophage receptor-mediated binding of neutrophil (leukocyte) elastase, I have studied the binding of leukocyte elastase and two other neutrophil granule glycoproteins, cathepsin G and lactoferrin, to human alveolar macrophages. Saturable binding of all three ligands at 0 degrees C was observed, with equilibrium dissociation constants of 4.0 x 10(-7), 2.0 x 10(-7), and 1.7 x 10(-6) M, respectively. All bound to a similar number (54-73 x 10(6)) of sites per cell. Binding of all three ligands was inhibited by the polysaccharide fucoidin, and extensive cross-inhibition of their binding to macrophages was observed. The results indicate that alveolar macrophages possess a relatively low-affinity, high-volume receptor for a family of neutrophil granule glycoproteins, which would be ideally suited for clearing released neutrophil granule contents from the extracellular space in inflamed tissues.     

Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937

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.