Specific Granule Protein Research Articles

An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma

hCAP-18 is a newly described protein of human neutrophilic granulocytes which belongs to the cathelicidin family of antimicrobial proteins. Members of this protein family share a common N-terminal sequence followed by a highly diverse antimicrobial, cationic C-terminus. The present work describes the production of recombinant hCAP-18, the generation of antibodies to the protein and the development of an accurate, sensitive and specific ELISA for the detection of hCAP-18 in cells, plasma and urine with a detection limit of 0.084 ng/ml. The amount of hCAP-18 in neutrophils is 0.627 μg protein per 10 6 cells. The plasma level is 1.18 μg/ml which is several fold higher than for other neutrophil specific granule proteins. hCAP-18 is present in plasma as high molecular weight complexes. In accordance with this, hCAP-18 is barely excreted in the urine. The bone marrow appears to be the major source of plasma hCAP-18. The high level of hCAP-18 in plasma may provide an important defense against microorganisms and endotoxins.

Sorting of the Specific Granule Protein, NGAL, During Granulocytic Maturation of HL-60 Cells

AbstractThe different types of human neutrophil granules (azurophil, specific, and gelatinase granules) are formed sequentially during maturation of neutrophils from the promyelocyte stage to the band cell stage. The promyelocytic HL-60 cells can maturate to segmented granulocytes but are incapable of activating the transcription of any known intragranular protein, normally located in specific or gelatinase granules. To study the sorting of granule proteins during maturation, we transfected HL-60 cells with the specific granule protein NGAL, inserted under control of a cytomegalovirus promoter. We previously showed that NGAL is sorted to azurophil granules and colocalizes with myeloperoxidase in undifferentiated HL-60 cells. We show here that, when such transfected HL-60 cells differentiate into granulocytes, newly synthesized NGAL is not retained in granules but is constitutively secreted. This indicates that highly specific mechanisms must exist that are responsible for diverting transport vesicles into storage granules, and that HL-60 cells not only lack the ability to activate transcription of specific granule proteins, but also lose the ability to form storage granules during maturation.

Ultrastructural localization of Charcot-Leyden crystal protein in human eosinophils and basophils.

The Charcot-Leyden crystal (CLC) protein with lysophospholipase activity and carbohydrate-binding properties is a characteristic constituent of eosinophils and basophils. We investigated its subcellular distribution using immunoelectron microscopy. Eosinophil progenitors, mature eosinophils and basophils all contained CLC protein in their cytosol and in the euchromatin of the nucleus. A minor population of granules in eosinophils, increasing in number with maturation, and a more abundant granule-population in basophils, were found to contain CLC protein. Double-labeling experiments showed, in eosinophils, that CLC protein-containing granules contain also eosinophil peroxidase, a characteristic specific granule protein. This suggests a relationship between the CLC protein-containing organelle and the specific granule. In basophils both the CLC protein positive and the negative granules showed the same characteristic particulate-like structure of the granular matrix and both share the same membrane marker CD63. In nasal polyps, macrophages were observed phagocytosing necrotic eosinophils. In these macrophages CLC protein-containing vesicles were observed, probably representing late endosomes. The dual (cytosolic/nuclear and granular) localization of CLC protein suggests that this protein enters both a secretory and a nonsecretory pathway during its biosynthesis, indicating functional roles for this protein both within the cell and extracellularly.

Targeting of proteins to granule subsets is determined by timing and not by sorting: The specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells.

The mechanism of protein targeting to individual granules in cells that contain different subsets of storage granules is poorly understood. The neutrophil contains two highly distinct major types of granules, the peroxidase positive (azurophil) granules and the peroxidase negative (specific and gelatinase) granules. We hypothesized that targeting of proteins to individual granule subsets may be determined by the stage of maturation of the cell, at which the granule proteins are synthesized, rather than by individual sorting information present in the proteins. This was tested by transfecting the cDNA of the specific granule protein, NGAL, which is normally synthesized in metamyelocytes, into the promyelocytic cell line HL-60, which is developmentally arrested at the stage of formation of azurophil granules, and thus does not contain specific and gelatinase granules. Controlled by a cytomegalovirus promoter, NGAL was constitutively expressed in transfected HL-60 cells. This resulted in the targeting of NGAL to azurophil granules as demonstrated by colocalization of NGAL with myeloperoxidase, visualized by immunoelectron microscopy. This shows that targeting of proteins into distinct granule subsets may be determined solely by the time of their biosynthesis and does not depend on individual sorting information present in the proteins.

SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis-specific gene product and to a rodent sperm-coating glycoprotein

A novel 28 kDa glycoprotein was purified from exocytosed material from human neutrophils and its primary structure partially determined. Degenerate oligonucleotide primers were used to amplify cDNA clones from a human bone marrow cDNA library. The deduced 245 amino acid sequence of the 2124 bp full-length cDNA showed high degrees of similarity to the deduced sequences of the human gene TPX-1 and of sperm coating glycoprotein from rat and mouse. Subcellular fractionation of human neutrophils indicated that the protein is localized in specific granules. The protein was named SGP28 (specific granule protein of 28 kDa).

HCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules

A 19 kDa protein was identified in specific granules of human neutrophils. A full-length cDNA clone was isolated from a human CML cDNA library, based on amino-acid sequences of isolated tryptic fragments. This clone includes the recently identified cDNA for FALL-39/CAP-18. Aminoacid sequences of proteolytic fragments derived both from the conserved N-terminal cathelin-like region and the highly variable C-terminal region characteristic of this family of bactericidal, LPS binding proteins, were in complete agreement with the sequence deduced from the cDNA. Thus, the 19 kDa protein is hCAP-18, stored as a ‘pro-peptide’ in specific granules.

Sputum peroxidase activity correlates with the severity of lung disease in cystic fibrosis.

Patients with cystic fibrosis (CF) of the same age differ significantly in their degree of pulmonary disease. Based on preliminary observations, we postulated that the activity of myeloperoxidase would be significantly increased in patients with greater structural lung damage than in those with less lung damage. Acid extracts of weighed sputum samples were assayed for lactoferrin concentrations by ELISA. Activities of peroxidase, cathespsin G, and elastase (with and without proteinase 3) were determined by kinetic analysis using chromogenic substrates. The patients were divided into quartiles based on their Brasfield chest-radiograph score. Patients in the first quartile (least amount of structural lung abnormality) were compared to those in the fourth quartile. The concentration of lactoferrin, a specific (secondary) granule protein of neutrophils, did not differ between the two patient groups. However, the activities of the neutrophil primary granule proteins, peroxidase, elastase, and elastase plus proteinase 3, were significantly elevated in the group with the most structural lung abnormality. Sputum albumin concentration was used to estimate leakages of plasma proteins into the airways. Peroxidase activity, but not the activity of cathepsin G, of elastase, or of elastase plus proteinase 3, correlated significantly with albumin/g sputum in both quartile groups. To confirm the association of sputum peroxidase activity with differences in lung structure and to test its correlation with lung function, spirometry was performed in a second group of patients during the week prior to the time of sputum sampling. In this second group, increased sputum peroxidase activity was associated with worse Brasfield scores and with decreased percent-predicted forced expiratory volume in 1 sec.(ABSTRACT TRUNCATED AT 250 WORDS)

HOCI production by human neutrophils activated by surface-associated IgG: Requirement for influx of extracellular calcium

Neutrophils produce large quantities of HOCl when stimulated by surface-associated immunoglobulin G, a result not seen when neutrophils are stimulated with soluble complexes of IgG. Compared with unactivated cells or cells stimulated with soluble aggregates of IgG, a significant influx of extracellular 45Ca2+ was observed in cells activated by surface-associated IgG. Removal of extracellular calcium with EGTA almost completely blocked HOCl production. Similarly, treatment of neutrophils with lanthanum, which has been shown to interfere with calcium channels, also effectively blocked HOCl production. These results were not secondary to an overall decrease in activation, as superoxide production and release of the specific granule protein lactoferrin and the azurophilic granule protein myeloperoxidase were not significantly altered by lanthanum or EGTA. Production of H2O2, the precursor of HOCl, was similarly decreased by both EGTA and lanthanum. Induction of extracellular calcium influx with a calcium ionophore in the presence of soluble aggregates of IgG resulted in HOCl production. Production of HOCl is not sensitive to inhibition by pretreatment of cells with pertussis toxin. These observations indicate that the differences in the biological responses of human neutrophils to surface-associated IgG compared with soluble aggregates of IgG are associated with differing signaling events, including influx of extracellular calcium.

Phosphatidic acid induces the release of beta-glucuronidase but not lactoferrin from electropermeabilized human neutrophils.

We studied the degranulation reaction of electropermeabilized human neutrophils induced by 1,2-didecanoyl-3-sn-phosphatidic acid (PA10). PA10 dose-dependently induced the release of beta-glucuronidase, an enzyme of azurophil granules, but did not induce the release of lactoferrin, a protein of specific granules. The enzyme release by PA10 absolutely required Ca2+, ATP, and Mg2+ and the concentrations for the half-maximal response were 2.5 microM, 60 microM, and 0.25 mM, respectively. Although Ca2+ alone at concentrations higher than 10 microM induced the release of both beta-glucuronidase and lactoferrin, the extents of the release were far less than that of the beta-glucuronidase release by PA10. Phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol induced the release of lactoferrin alone at concentrations of Ca2+ below 0.5 microM while they induced the release of both beta-glucuronidase and lactoferrin at higher Ca2+ concentrations, indicating that the degranulation induced by PA10 is not mediated by diacylglycerol which might be formed from PA. The degranulation reactions induced by PA10 and PMA were dose-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors, although no direct activation of protein kinase C by PA10 was observed. The extent of the beta-glucuronidase release by PA10 was not enhanced by the addition of PMA. Propranolol, which inhibits protein kinase C as well as phosphatidic acid phosphohydrolase, strongly inhibited the degranulation reactions induced by PA10 and PMA. Ethanol, a metabolic modulator of phospholipase D, and cyclic AMP did not affect the degranulation reactions by PMA and PA10.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils

Neutrophil gelatinase-associated lipocalin (NGAL) is a novel 25-kD protein of human neutrophils, that is in part covalently complexed with neutrophil gelatinase. However, both NGAL and gelatinase exist mainly in forms not associated with each other. An explanation for this phenomenon might be that the unassociated proteins reside in different subcellular compartments. The aim of the present study was to determine the subcellular localization of NGAL. An enzyme-linked immunosorbent assay (ELISA) for NGAL was developed using specific anti-NGAL antibodies. The assay was applied on subcellular fractions of neutrophils obtained after centrifugation of a postnuclear supernatant on a two-layer Percoll gradient. The distribution profile of NGAL was found to colocalize strictly with the distribution profile of lactoferrin. This was confirmed by immunogold double-labeling of frozen thin sections of neutrophils that showed a high degree of colocalization of NGAL and lactoferrin, and by exocytosis experiments, which showed lactoferrin, vitamin B12-binding protein, and NGAL to be similarly released upon stimulation. Therefore, NGAL is a novel matrix protein of specific granules and thus partly segregated from gelatinase, the major part of which is located in a separate compartment, the gelatinase granules. An ELISA specific for the NGAL/gelatinase complex was developed and the subcellular distribution and release of this complex was determined. The distribution and mobilization of the complex allowed us to confirm the existence of differentially mobilized granule subpopulations among peroxidase negative granules.

Identification of Neutrophil Gelatinase-Associated Lipocalin as a Novel Matrix Protein of Specific Granules in Human Neutrophils

AbstractNeutrophil gelatinase-associated lipocalin (NGAL) is a novel 25-kD protein of human neutrophils, that is in part covalently complexed with neutrophil gelatinase. However, both NGAL and gelatinase exist mainly in forms not associated with each other. An explanation for this phenomenon might be that the unassociated proteins reside in different subcellular compartments. The aim of the present study was to determine the subcellular localization of NGAL. An enzyme-linked immunosorbent assay (ELISA) for NGAL was developed using specific anti-NGAL antibodies. The assay was applied on subcellular fractions of neutrophils obtained after centrifugation of a postnuclear supernatant on a two-layer Percoll gradient. The distribution profile of NGAL was found to colocalize strictly with the distribution profile of lactoferrin. This was confirmed by immunogold double-labeling of frozen thin sections of neutrophils that showed a high degree of colocalization of NGAL and lactoferrin, and by exocytosis experiments, which showed lactoferrin, vitamin B12-binding protein, and NGAL to be similarly released upon stimulation. Therefore, NGAL is a novel matrix protein of specific granules and thus partly segregated from gelatinase, the major part of which is located in a separate compartment, the gelatinase granules. An ELISA specific for the NGAL/gelatinase complex was developed and the subcellular distribution and release of this complex was determined. The distribution and mobilization of the complex allowed us to confirm the existence of differentially mobilized granule subpopulations among peroxidase negative granules.

Eosinophil cationic granule proteins impair thrombomodulin function. A potential mechanism for thromboembolism in hypereosinophilic heart disease.

Thromboembolism is a prominent but poorly understood feature of eosinophilic, or Loeffler's endocarditis. Eosinophil (EO) specific granule proteins, in particular major basic protein (MBP), accumulate on endocardial surfaces in the course of this disease. We hypothesized that these unusually cationic proteins promote thrombosis by binding to the anionic endothelial protein thrombomodulin (TM) and impairing its anticoagulant activities. We find that MBP potently (IC50 of 1-2 microM) inhibits the capacity of endothelial cell surface TM to generate the natural anticoagulant activated protein C (APC). MBP also inhibits APC generation by purified soluble rabbit TM with an IC50 of 100 nM without altering its apparent Kd for thrombin or Km for protein C. This inhibition is reversed by polyanions such as chondroitin sulfate E and heparin. A TM polypeptide fragment comprising the extracellular domain that includes its naturally occurring anionic glycosaminoglycan (GAG) moiety (TMD-105) is strongly inhibited by MBP, whereas its counterpart lacking the GAG moiety (TMD-75) is not. MBP also curtails the capacity of TMD-105 but not TMD-75 to prolong the thrombin clotting time. Thus, EO cationic proteins potently inhibit anticoagulant activities of the glycosylated form of TM, thereby suggesting a potential mechanism for thromboembolism in hypereosinophilic heart disease.

Neutrophil and eosinophil involvement of the small bowel in patients with celiac disease and Crohn's disease: Studies on the secretion rate and immunohistochemical localization of granulocyte granule constituents

purpose: The concentrations of myeloperoxidase (MPO), a neutrophil granule constituent, and eosinophil cationic protein (ECP), a specific eosinophil granule protein, were measured in jejunal perfusion fluid in an attempt to elucidate the neutrophil and eosinophil involvement of the small bowel in health and disease. patients and methods: The control group consisted of 14 males and two females. Ten patients (seven males and three females) with Crohn's disease and seven patients (two males and five females) with celiac disease were also studied; in addition, one patient with relapsing giardiasis, one patient with giardiasis and complete absence of plasma cells in small intestinal lamina propria, and one patient with selective IgA deficiency and no IgA plasma cells in duodeno-jejunal lamina propria were evaluated. Segmental perfusion of the jejunum was performed according to a previously described method. MPO and ECP were measured by radioimmunoassays. results: In healthy control subjects, the concentrations of both granule proteins were in a narrow range and much higher than would have been anticipated from passive leakage from circulating blood. In patients with celiac disease, the perfusion fluid concentrations of MPO and ECP were on average 3.5 and eight times, respectively, higher than the values seen in the controls. The jejunal segment perfused in patients with Crohn's disease was endoscopically and histologically normal. The perfusion fluid concentrations of MPO and ECP were increased 3.5 and two times, respectively, compared with that in the control subjects. Both patient groups and the control group had similar perfusion fluid concentrations of albumin. Data on MPO and ECP expressed as jejunal secretion rates gave the same differences between patients and controls as just described for the jejunal fluid concentrations. Immunohistochemical studies of jejunal biopsy specimens from another group of patients with celiac disease demonstrated a prominent extracellular deposit of ECP in the lamina propria of the atrophic intestinal mucosa, whereas the release of neutrophil constituents (cathepsin G, MPO) was scarce. In Crohn's disease, an extracellular degranulation ECP and, to a lesser extent, of cathepsin G was observed in relation to ulcerations only. conclusion: Data obtained indicate that the local release of neutrophil and eosinophil granule components is enhanced in the jejunal tissue from patients with celiac sprue and Crohn's disease. The prominent extracellular deposit of eosinophil granule constituents with cytotoxic properties at the site of inflammatory intestinal lesions in celiac sprue might reflect a pathophysiologic mechanism. The deposit of eosinophil and neutrophil granule components in ulcerated areas in Crohn's disease suggests that granulocytes may contribute to the inflammatory lesions also in this disease.

The neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules.

The glycoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove absorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils.

Induction of neutrophil enzyme release by rheumatoid factors: Evidence for differences based on molecular characteristics

Cathepsin G and elastase are two neutrophil proteases capable of degrading the major structural macromolecules of the joint. Evaluation of factors capable of inducing the release of these enzymes is crucial to the understanding of neutrophil-mediated tissue destruction. We have evaluated the effects of IgM rheumatoid factor (RF), as well as monomeric and polymeric forms of IgA RF, on the release of neutrophil elastase, cathepsin G, and the specific granule protein lactoferrin. None of these rheumatoid factors alone was able to induce more lysosomal protein release than media controls. Under conditions used in this study, aggregated human IgG was able to induce slightly more release than media controls. The addition of IgM RF or polymeric IgA RF to the aggregated IgG resulted in release of significantly more lysosomal proteins than aggregates alone. In contrast, monomeric IgA RF, even in the presence of aggregated IgG, was unable to augment enzyme release. These results suggest that differences in the molecular characteristics of RF found in synovial fluid may significantly influence the contribution of RF to tissue injury in rheumatoid arthritis.

Purified plasma factor XIIa aggregates human neutrophils and causes degranulation

Plasma kallikrein has been shown to aggregate human neutrophils and release human neutrophil elastase. However, neutrophils resuspended in factor XII-deficient plasma released only 30% of the elastase compared with normal plasma. Isolated human neutrophils were aggregated in a concentration-dependent fashion by 0.06 to 0.6 U/mL factor XIIa (0.022 to 0.22 mumol/L). Factor XIIa (0.1 to 1.0 U/mL) also induced neutrophil degranulation as evidenced by a concentration-dependent release of the specific granule protein, lactoferrin, and azurophilic granule protease, elastase. The release of neutrophil elastase was biphasic, reaching 40% of maximum at 15 seconds with maximal release by 90 minutes. The active site of factor XIIa was required, since the synthetic inhibitor, D-Pro-Phe-Arg-CH2Cl, which reacts with an essential histidine, and the natural plasma inhibitor, Cl-inhibitor, which interacts with the critical serine, both inhibit by more than 90% the release of elastase. The heavy chain is also required, since factor XII fragments failed to aggregate neutrophils or stimulate degranulation. Factor XIIa (0.6 U/mL) can completely correct the defect in elastase release evident in factor XII-deficient plasma. These studies demonstrate that factor XIIa, at concentrations potentially obtainable in plasma in disease states, can activate neutrophils, and thus may participate in the inflammatory response.

Purified Plasma Factor XHa Aggregates Human Neutrophils and Causes Degranulation

Plasma kallikrein has been shown to aggregate human neutrophils and release human neutrophil elastase. However, neutrophils resuspended in factor XII-deficient plasma released only 30% of the elastase compared with normal plasma. Isolated human neutrophils were aggregated in a concentration-dependent fashion by 0.06 to 0.6 U/mL factor XIIa (0.022 to 0.22 mumol/L). Factor XIIa (0.1 to 1.0 U/mL) also induced neutrophil degranulation as evidenced by a concentration-dependent release of the specific granule protein, lactoferrin, and azurophilic granule protease, elastase. The release of neutrophil elastase was biphasic, reaching 40% of maximum at 15 seconds with maximal release by 90 minutes. The active site of factor XIIa was required, since the synthetic inhibitor, D-Pro-Phe-Arg-CH2Cl, which reacts with an essential histidine, and the natural plasma inhibitor, Cl-inhibitor, which interacts with the critical serine, both inhibit by more than 90% the release of elastase. The heavy chain is also required, since factor XII fragments failed to aggregate neutrophils or stimulate degranulation. Factor XIIa (0.6 U/mL) can completely correct the defect in elastase release evident in factor XII-deficient plasma. These studies demonstrate that factor XIIa, at concentrations potentially obtainable in plasma in disease states, can activate neutrophils, and thus may participate in the inflammatory response.

Identification and characterization of calmodulin-binding proteins in islet secretion granules.

The binding of 125I-calmodulin to intact secretion granules and protein gel blots of secretion granules from pancreatic islet tissue was examined. Binding of 125I-calmodulin to intact secretion granules was Ca2+-dependent and inhibited by the calmodulin inhibitors trifluoperazine and calmidazolium. Binding was inhibited by excess (200 nM) unlabeled calmodulin, but not by parvalbumin, a Ca2+-binding protein which has little sequence homology to calmodulin. In order to study the binding of calmodulin to specific secretion granule proteins, secretion granules were solubilized, and the solubilized proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose, and incubated with 125I-calmodulin. Autoradiograms of the protein gel blots revealed the presence of three major calmodulin-binding proteins with approximate molecular weights of 73,000, 64,000, and 58,000. These proteins reversibly bound calmodulin in a calcium-dependent manner. Unlabeled calmodulin in the range of 0.1-1.0 nM competed with 125I-calmodulin for binding to these proteins, whereas troponin and parvalbumin were 100 and 1000-fold less effective, respectively. Trifluoperazine blocked binding to the granule proteins in a range of 10(-4) to 10(-5) M, and calmidazolium was effective between 10(-5) and 10(-6) M. Trypsin, at a concentration which did not lyse granules, markedly inhibited calmodulin binding to intact secretion granules. Protein blots from trypsin-treated granules showed that the three major calmodulin-binding proteins were absent. These results indicate that Ca2+-dependent calmodulin-binding proteins are present on the cytoplasmic surface of islet secretion granules and are consistent with the hypothesis that these proteins may play a role in secretion granule exocytosis.

Eosinophil cationic protein in inflammatory synovial effusions as evidence of eosinophil involvement.

Eosinophils are seldom noted in inflammatory synovial fluids but are reported to infiltrate the synovial tissue in inflammatory arthritides. To elucidate a possible role for eosinophils in inflammatory joint reactions the concentrations of eosinophil cationic protein (ECP)--a specific granule protein from eosinophils--were measured by radioimmunoassay in 90 synovial fluids from patients with various inflammatory arthritides (rheumatoid arthritis, reactive and crystal arthritides, Reiter's disease and psoriatic arthropathy). In the same specimens lactoferrin was measured as an indicator of neutrophil-involved inflammation. In comparison with the normal circulating levels of ECP and lactoferrin the measured synovial fluid concentrations of both proteins were considerably raised in all patient groups with inflammatory joint diseases in contrast to patients with non-inflammatory arthritides. There was a striking positive correlation between the ECP and lactoferrin synovial fluid concentrations. These data indicate that eosinophil activation is prominent in inflammatory joint reactions and is linked to the activation of neutrophils. The regulation of degranulation or secretion by eosinophils is unknown. Our in-vitro studies showed that peripheral blood isolated neutrophils as well as eosinophils degranulated when exposed to IgG complexes. However, eosinophil degranulation was modest compared with neutrophil degranulation. These data suggest that neutrophil phagocytosis of, for example, immune complexes may be one major mechanism in neutrophil degranulation but that other factors determine the appearance of eosinophil products in inflammatory synovial effusions. The possible modulatory or harmful role of eosinophils in inflammatory joint disease can at present only be speculated on.

Signs of neutrophil and eosinophil activation in adult respiratory distress syndrome.

Circulating levels of lactoferrin, a specific granule protein of neutrophilic leukocytes, and eosinophil cationic protein (ECP), a specific granule protein of eosinophilic leukocytes, were serially measured in 19 patients at risk for adult respiratory distress syndrome (ARDS). Those patients who developed ARDS had significantly higher concentrations of both proteins than the patients without signs of ARDS. High ECP levels were observed in spite of peripheral eosinopenia. The lactoferrin levels were also increased in relation to circulating numbers of neutrophils. These findings are consistent with an enhanced turnover and/or activity of eosinophils and neutrophils in ARDS and thereby support other clinical and experimental observations suggesting a central pathophysiologic role for granulocytes in ARDS. No relation was found between ARDS or serum concentrations of lactoferrin or ECP and degree of complement consumption, suggesting that other mechanisms besides complement activation may underlie granulocyte activation in ARDS.