Rapid cDNA library screening using the polymerase chain reaction.
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Base Sequence↗
Biomedical subjects
S J Ackerman
Publications and source records attributed to S J Ackerman.
Eosinophil degranulation. An immunologic determinant in the pathogenesis of the Mazzotti reaction in human onchocerciasis.
Onchocerciasis patients treated with diethylcarbamazine often undergo a severe inflammatory response, the Mazzotti reaction. To assess the eosinophil's role in the pathogenesis of the Mazzotti reaction, we obtained serial blood, plasma, and skin biopsy specimens from 21 heavily infected patients and 3 endemic controls, both before and during therapy with diethylcarbamazine. Samples were analyzed for blood eosinophils, plasma levels of eosinophil granule major basic protein (MBP) and eosinophil-derived neurotoxin, eosinophil infiltration and eosinophil and mast cell degranulation in the skin. After the first dose of diethylcarbamazine, blood eosinophils fell from a pre-treatment level of 888 +/- 111 to 203 +/- 42 cells/mm3 at 8 h. This decrease was followed by a marked eosinophilia developing over the remaining 7 days of treatment and 14 days of follow-up. Plasma eosinophil-derived neurotoxin levels increased from 56 +/- 4 ng/ml pretreatment to a peak of 82 +/- 9 ng/ml at 8 h and returned to pretreatment levels by 48 h. Beginning at 12 h, plasma MBP levels increased from 730 +/- 74 ng/ml pretreatment to a peak of 1140 +/- 74 ng/ml after 5 days. Pretreatment skin biopsies stained for MBP by immunofluorescence showed a bright fibrillar pattern in the dermis consistent with chronic eosinophil degranulation; the MBP was localized on elastic tissue fibers. After treatment, skin biopsy specimens showed both the pretreatment fibrillar MBP staining pattern as well as focal eosinophil degranulation. Deposition of MBP around microfilariae in the papillary dermis was visible as early as 1.5 h. The lowest blood eosinophil levels and peak plasma eosinophil-derived neurotoxin levels coincided with the infiltration and degranulation of eosinophils in the skin. Mast cell degranulation in the skin was maximal by the first posttreatment biopsy (1.5 h) coincident with the beginning of eosinophil degranulation. Although the pathogenesis of the Mazzotti reaction is clearly complex, our results indicate that eosinophil degranulation is characteristic of the response and that it occurs with a time course suggestive of a role for the eosinophil in determining the clinical and pathologic manifestations of the reaction.
Adult↗
Ultrastructural localization of Charcot-Leyden crystal protein (lysophospholipase) and peroxidase in macrophages, eosinophils, and extracellular matrix of the skin in the hypereosinophilic syndrome.
Electron microscopy, ultrastructural cytochemistry, and postembedding immunogold ultrastructural immunocytochemistry were used to study a papular cutaneous lesion from a patient with the hypereosinophilic syndrome. Peroxidase activity was detected cytochemically in 40-microns sections of skin utilizing the substrate diaminobenzidine; Charcot-Leyden crystal (CLC) protein was detected immunocytochemically in skin utilizing a postembedding immunogold technique; and a combined method was used where postembedding immunogold staining of CLC protein was performed on sections previously prepared to detect peroxidase activity. We describe a unique, eosinophil-rich inflammatory process in involved skin which contained extraordinary numbers of morphologically activated macrophages. Electron microscopy demonstrated (a) widespread eosinophil necrosis, (b) interstitial CLC formation, (c) macrophage activation, endocytosis, and phagocytosis, and (d) CLC formation in phagosomes of activated macrophages. Peroxidase activity was present as follows: (a) in the matrix of eosinophil specific granules in eosinophil cytoplasm, in membrane-bound specific granules released into interstitial tissues from dying eosinophils, being phagocytized by activated macrophages, and within macrophage phagosomes; (b) as amorphous interstitial debris; (c) in cytoplasm and nuclei of damaged eosinophils in the dermal tissues as well as in macrophage phagosomes; and (d) in endocytotic vesicles and vacuoles of macrophages and in CLC-containing phagosomes of macrophages. CLC protein was localized by immunocytochemistry to (a) eosinophil primary granules, (b) cytoplasm and nuclei of damaged eosinophils located in the interstitial tissues or within macrophage phagosomes, (c) CLC located in interstitial tissues adjacent to necrotic eosinophils and in macrophage phagosomes, and (d) aggregates of amorphous protein bound to macrophage surfaces; endocytotic vesicles and vacuoles of macrophages; amorphous protein aggregates in macrophage lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)
Biopsy↗
Ultrastructural localization of Charcot-Leyden crystal protein (lysophospholipase) to intracytoplasmic crystals in tumor cells of primary solid and papillary epithelial neoplasm of the pancreas.
Charcot-Leyden crystals (CLC), composed of a single protein with lysophospholipase activity, have been traditionally associated with eosinophil-rich disorders. Atypically shaped and typical CLC were noted by electron microscopy in the surgical sample from a patient with solid and papillary epithelial neoplasm of the pancreas. This rare primary tumor of the pancreas with limited aggressive behavior also contained damaged and partially or completely degranulated eosinophils in the tumor stroma. We localized CLC protein by ultrastructural immunogold staining to the CLC within vacuolar structures and in vesicles of tumor cell cytoplasm as well as to the cytoplasm and nucleus of eosinophils in the tumor stroma. These findings provide evidence that epithelial tumor cells contain Charcot-Leyden crystal protein that most likely originates from tumor stroma eosinophils. Tumor stroma eosinophils have generally been associated with improved prognoses in a wide variety of epithelial neoplasms. The role of tumor CLC protein (lysophospholipase) in these settings deserves further investigation.
Adult↗
An alternative method for labeling oligonucleotide probes for screening cDNA libraries.
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Gene Library↗
Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity.
We have isolated a 725-bp full-length cDNA clone for the human eosinophil cationic protein (ECP). ECP is a small, basic protein found in the matrix of the eosinophil's large specific granule that has cytotoxic, helminthotoxic, and ribonuclease activity, and is a member of the ribonuclease multigene family. The cDNA sequence shows 89% sequence identity with that reported for the related granule protein, eosinophil-derived neurotoxin (EDN). The open reading frame encodes a previously unidentified 27-amino acid leader sequence preceding a 133-residue mature ECP polypeptide with a molecular mass of 15.6 kD. The encoded amino acid sequence of ECP shows 66% identity to that of EDN and 31% identity to that of human pancreatic ribonuclease, including conservation of the essential structural cysteine and cataytic lysine and histidine residues. mRNA for ECP was detected in eosinophil-enriched peripheral granulocytes and in a subclone of the promyelocytic leukemia line, HL-60, induced toward eosinophilic differentiation with IL-5. No ECP mRNA was detected in uninduced HL-60 cells, or in HL-60 cells induced toward monocytic differentiation with vitamin D3 or toward neutrophilic differentiation with DMSO. In contrast, mRNA for EDN was detected in uninduced HL-60 cells and was upregulated in HL-60 cells induced with DMSO. Despite similarities in sequence and cellular localization, these results suggest that ECP and EDN are subject to different regulatory mechanisms.
Amino Acid Sequence↗
Molecular cloning of the human eosinophil-derived neurotoxin: a member of the ribonuclease gene family.
We have isolated a 725-base-pair cDNA clone for human eosinophil-derived neurotoxin (EDN). EDN is a distinct cationic protein of the eosinophil's large specific granule known primarily for its ability to induce ataxia, paralysis, and central nervous system cellular degeneration in experimental animals (Gordon phenomenon). The open reading frame encodes a 134-amino acid mature polypeptide with a molecular mass of 15.5 kDa and a 27-residue amino-terminal hydrophobic leader sequence. The sequence of the mature polypeptide is identical to that reported for human urinary ribonuclease [Beintema, J. J., Hofsteenge, J., Iwama, M., Morita, T., Ohgi, K., Irie, M., Sugiyama, R. H., Schieven, G. L., Dekker, C. A. & Glitz, D. G. (1988) Biochemistry 27, 4530-4538] and to the amino-terminal sequence of human liver ribonuclease [Sorrentino, S., Tucker, G. K. & Glitz, D. G. (1988) J. Biol. Chem. 263, 16125-16131]; the cDNA encodes a tryptophan in position 7, which was previously unidentified in the amino acid sequences of EDN or the urinary and liver ribonucleases. Both EDN and the related granule protein, eosinophil cationic protein, have ribonucleolytic activity; sequence similarities among EDN, eosinophil cationic protein, ribonucleases from liver, urine, and pancreas, and angiogenin define a ribonuclease multigene family. mRNA encoding EDN was detected in uninduced HL-60 cells and was up-regulated in cells induced toward eosinophilic differentiation with B-cell growth factor 2/interleukin 5 and toward neutrophilic differentiation with dimethyl sulfoxide. EDN mRNA was detected in mature neutrophils even though EDN-like neurotoxic activity is not found in neutrophil extracts. These results suggest that neutrophils contain a protein that is closely related or identical to EDN.
Amino Acid Sequence↗
Eosinophils and human disease.
The likely roles of the eosinophil leukocyte in human disease are reviewed. The eosinophil is richly endowed with toxic cationic proteins and is able to mount a respiratory burst. Thus, eosinophils have the capability to damage various targets, and evidence exists that they do so during helminth infections and during the course of many hypersensitivity diseases. Here we discuss the role of the eosinophil in human onchocerciasis with particular attention to the Mazzotti reaction. We also discuss other diseases where eosinophil degranulation is seen, especially cutaneous diseases. Finally, the possible role(s) of the granule major basic protein in human pregnancy is noted.
Animals↗
The new gestalt: asthma as a chronic inflammatory disease.
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Asthma↗
Cytoplasmic lipid bodies of human neutrophilic leukocytes.
The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids.
Arachidonic Acid↗
Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to granules and intragranular crystals in mature human basophils.
Human blood basophils have been shown to form Charcot-Leyden crystals (CLC) and to contain quantities of CLC protein comparable to the eosinophil (Ackerman SJ, Weil GJ, Gleich GJ, J Exp Med 155:1597, 1982). We examined the subcellular localization of CLC protein in human basophils from peripheral blood using an ultrastructural postembedding immunogold method and affinity chromatography-purified polyclonal primary antibody to purified human eosinophil CLC protein. We found CLC protein to be uniquely associated with the main, large, particle-filled granule population of human basophils and particularly within intragranular Charcot-Leyden crystals in these granules. Rarely, CLC protein was localized within small, smooth perigranular vesicles. Neutrophils, lymphocytes, and monocytes present in preparations stained for the CLC protein were negative as were controls for the specificity of the immunogold staining. Mature eosinophils contained small numbers of positive crystalloid-free primary granules, as previously reported (Dvorak AM, Letourneau L, Login GR, Weler PF, Ackerman SJ, Blood 72:150, 1988). The presence of CLC protein in hexagonal and pyramidal crystals regularly present in activated types II and III basophils suggests two possibilities currently being investigated. Like eosinophils, basophils could synthesize CLC protein, or eosinophil-derived CLC protein could be internalized and stored in basophil granules. Regardless of the mechanism for acquisition of CLC protein (lysophospholipase) by basophils, the identification and subcellular localization of this enzyme in basophils requires that it be considered in basophil cell biology as well as in the pathobiology of basophil-rich reactions.
Basophils↗
Crystallographic characterization of human eosinophil Charcot-Leyden crystals.
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Eosinophils↗
Eosinophil granule cationic proteins: major basic protein is distinct from the smaller subunit of eosinophil peroxidase.
Major basic protein and eosinophil peroxidase are predominant cationic proteins in the cytoplasmic granules of human eosinophilic leukocytes. Each of these proteins has been purified, and major basic protein and the lower molecular weight subunit of eosinophil peroxidase have been found to comigrate on polyacrylamide gel electrophoresis in sodium dodecyl sulfate with similar apparent molecular weights of about 14,700. Because previous molecular weight estimates for these proteins have not recognized the similar molecular weights of these two cationic eosinophil granule constituents, we have evaluated the possible relatedness of these proteins. Upon protein sequence analyses, it was found that the N-terminal 20 amino acid residues of each of these two purified polypeptides differed. These findings established that major basic protein and the smaller subunit of eosinophil peroxidase, although of comparable molecular weights, are two distinct proteins within the cytoplasmic granules of human eosinophils.
Amino Acid Sequence↗
Induction of interleukin-4-dependent IgE synthesis and interleukin-5-dependent eosinophil differentiation by supernatants of a human helper T-cell clone.
The human alloreactive T-cell clone A1 produces interleukin (IL)-4, IL-5, and granulocyte macrophage-colony stimulating factor (GM-CSF), but not IL-2 or interferon-gamma (IFN-gamma), as assessed by probing for mRNA transcripts, immunoassays, or bioassays. Supernatants from clone A1 induced IgE synthesis by normal human peripheral blood mononuclear cells. IL-4 was shown to be necessary and sufficient for the induction of IgE by clone A1 supernatants. In contrast, IgE induction by clone A1 supernatants and recombinant (r) IL-4 was inhibited by IFN-gamma. This suggests that the absence of IFN-gamma from the IL-4-containing A1 supernatants was important for their IgE-inducing ability. Supernatants from clone A1 could also specifically induce the growth of cord blood cell progenitors and their differentiation into eosinophils but not into basophils. rIL-5, but not rIL-4 or rGM-CSF, also induced eosinophil maturation. These data suggest that IL-5 secreted by clone A1 was responsible for its ability to induce eosinophil differentiation. The implications of the concomitant production of IL-4 and IL-5 by a single T-cell clone are discussed.
Cell Differentiation↗
Heterogeneity of leukotriene C4 production by eosinophils from asthmatic and from normal subjects.
We evaluated the formation of leukotriene C4 (LTC4) by peripheral blood eosinophils of different densities obtained from asthmatic and normal subjects. When stimulated with 1 microgram/ml of the calcium ionophore A23187 for 15 min at 37 degrees C, eosinophils with densities greater than 1.093 g/ml from asthmatic and normal subjects released 19.1 +/- 4.2 ng LTC4/10(6) eosinophils and 23.9 +/- 5.0 ng LTC4/10(6) eosinophils, respectively. In contrast, lower density eosinophils (densities 1.093 g/ml or less) isolated from the asthmatic subjects released significantly less LTC4 than did eosinophils of similar densities from normal subjects (41.6 +/- 3.0 versus 79.0 +/- 6.7 ng LTC4/10(6) eosinophils, p less than 0.05). Differences could not be demonstrated between the two subject groups in LTC4 metabolism, time course of extracellular release of LTC4, or dose response to A23187, nor were interactions between eosinophils and neutrophils with regard to LTC4 release evident. Thus, hypodense eosinophils elaborate greater quantities of LTC4 than do eosinophils of normal density whether obtained from normal or asthmatic subjects. However, the finding that peripheral blood eosinophils from asthmatic subjects have decreased capacity for the synthesis of LTC4 compared with cells of similar densities isolated from normal subjects demonstrates that the capacity of eosinophils to produce LTC4 is regulated by factors that are not necessarily reflected in the cell density.
Asthma↗
Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to a distinct crystalloid-free granule population in mature human eosinophils.
The Charcot-Leyden crystal (CLC) protein is a unique constituent of eosinophils and basophils. This protein forms the hexagonal bipyramidal crystals observed in tissues at sites of eosinophil accumulations, possesses lysophospholipase activity (lysolecithin acylhydrolase E.C.3.1.1.5), and comprises an estimated 7% to 10% of total eosinophil protein. The ultrastructural localization of CLC protein was studied in mature peripheral blood eosinophils from normal donors and from patients with the idiopathic hypereosinophilic syndrome. Subcellular localization was evaluated by immunoelectron microscopy using eosinophils, both from buffy coat and purified cell suspensions, that were fixed by a variety of methods. Immunochemical detection of CLC protein employed rabbit antiserum to eosinophil CLC protein, affinity chromatography-purified monospecific IgG antibodies, and postembedding immunogold techniques. Controls for specificity included (1) omission of the primary antibody to CLC protein and (2) substitution of primary antibody with a nonimmune preimmunization serum, a protein A-purified nonimmune IgG, or a protein A-purified nonreactive IgG prepared from solid-phase CLC protein-Sepharose-absorbed anti-CLC antiserum. CLC protein was localized to a minor (approximately 5%) subpopulation of eosinophil granules. These membrane-bound cytoplasmic granules were large (greater than 0.5 mu), were devoid of crystalloid inclusions, and were morphologically compatible with persisting eosinophil primary granules. The crystalloid-containing, large, specific granules did not stain for CLC protein. Insufficient numbers of small dense granules, lipid bodies, and vesiculotubular structures were present to adequately evaluate their potential as additional sites for the subcellular localization of CLC protein. The cellular specificity of the immunogold localization of CLC protein in the eosinophil was affirmed by the absence of staining in neutrophils and lymphocytes present in the same sections. The ultrastructural immunogold localization of CLC protein (lysophospholipase) to a large, crystalloid-free granule in mature circulating eosinophils supports the persistence of a distinct "primary" granule population that serves as a major intracytoplasmic repository for this enzyme.
Crystallization↗
Deposits of eosinophil granule proteins in cardiac tissues of patients with eosinophilic endomyocardial disease.
Eosinophilic endomyocardial disease is a complication of the hypereosinophilic syndrome and of several other disorders associated with high blood eosinophil counts. Eosinophil granule proteins may be involved in the development of these lesions. This multi-centre study investigated whether these proteins could be demonstrated within the cardiac tissues of eighteen patients with eosinophilic endomyocardial disease. Serial sections of tissue taken at necropsy or at cardiac biopsy were stained for eosinophil major basic protein by indirect immunofluorescence and for eosinophil cationic protein, eosinophil protein-X, and activated eosinophils by means of alkaline-phosphatase-linked monoclonal antibodies. Activated eosinophils and secreted eosinophil granule proteins were most evident within the necrotic and later stage thrombotic lesions and were found mainly within the areas of acute tissue damage in the endocardium and in the walls of small blood vessels. These findings suggest that eosinophil granule proteins are involved in cardiac injury, producing muscle damage and vascular injury which lead to the development of endomyocardial fibrosis.
Acute Disease↗
Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells.
Human eosinophils contain several distinctive proteins including eosinophil granule MBP and the membrane-associated CLC protein (lysophospholipase). Human basophils also contain these proteins, indicating biochemical similarities between eosinophils and basophils. To determine whether MBP or CLC protein is present in connective tissue mast cells, we studied human lung and cutaneous mast cells by immunofluorescence by utilizing specific antibodies to CLC and MBP. Cytocentrifuge slides of enriched lung mast cells and mast cells in sections of formalin-fixed, paraffin-embedded cutaneous tissue from urticaria pigmentosa lesions were stained for CLC and MBP. Neither pulmonary nor cutaneous mast cells stained for CLC protein or MBP. In contrast, lung and cutaneous eosinophils in the same preparations showed bright staining for both proteins. The failure to find CLC protein and MBP in mast cells provides additional evidence of dissimilarity between mast cells and basophils, and an immunochemical means to distinguish between them.
Basophils↗