The eosinophil cationic protein (ECP)' is one of several small, distinct argininerich proteins that have been isolated from the eosinophil's large specific granule. The molecular mass of human ECP has been estimated at 18-21 kD (1-3), depending on the degree of glycosylation (3). ECP has been shown to possess a wide variety of biological activities, including the ability to stimulate factor XII-dependent coagulation pathways (4), to neutralize the anticoagulant effects of heparin (5), and to inhibit lymphocyte proliferation induced by PHA or mixed lymphocyte reactions (6) . ECP is a potent cytotoxin; ECP-containing supernatants of activated eosinophils have been shown to be toxic to isolated myocardial cells in vitro (7), and both ECP and the eosinophil granule major basic protein (MBP) were found in the endothelial and endomyocardial lesions characteristic of the hypereosinophilic syndrome (8). Furthermore, ECP is also a potent helminthotoxin; destruction of schistosomula of Schistosoma mansont was reported at concentrations as low as 10' M (8-10-fold more active than MBP) (9, 10) . ECP has also been shown to kill trypomastigote and amastigote stages of Trypanosoma cruzi (11) . Both ECP and the related granule protein, eosinophil-derived neurotoxin (EDN), induce the neurotoxic effect known as the Gordon phenomenon (12)

The role(s) of the eosinophil Charcot-Leyden crystal (CLC) protein in eosinophil or basophil function or associated inflammatory processes is yet to be established. Although the CLC protein has been reported to exhibit weak lysophospholipase activity, it shows virtually no sequence homology to any known member of this family of enzymes. The X-ray crystal structure of the CLC protein is very similar to the structure of the galectins, members of a beta-galactoside-specific animal lectin family, including a partially conserved galectin carbohydrate recognition domain (CRD). In the absence of any known natural carbohydrate ligand for this protein, the functional role of the CLC protein (galectin-10) has remained speculative. Here we describe structural studies on the carbohydrate binding properties of the CLC protein and report the first structure of a carbohydrate in complex with the protein. Interestingly, the CLC protein demonstrates no affinity for beta-galactosides and binds mannose in a manner very different from those of other related galectins that have been shown to bind lactosamine. The partial conservation of residues involved in carbohydrate binding led to significant changes in the topology and chemical nature of the CRD, and has implications for carbohydrate recognition by the CLC protein in vivo and its functional role in the biology of inflammation.

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Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

We have recently shown that a proportion of previously designated human eosinophil “(Eo)-type” colonies in methylcellulose contain basophils and histamine (Denburg et al Blood 61:775, 1983). In the present studies, individual Eo-type colonies have been analyzed by cell morphology as well as by biochemical assays for histamine, Charcot- Leyden crystal protein (CLC), and eosinophil granule major basic protein (MBP). Clonal origin of single Eo-type colonies was confirmed by G6PD isoenzyme analysis. Morphological observations of such colonies revealed the existence of two distinct colony types: (1) Eo type containing 100% basophils and (2) Eo type containing mixtures of basophils and eosinophils, including cells with mixed basophil- eosinophil granulation. Histamine was not detected in pure, mature peripheral blood eosinophils. Immunofluorescent studies demonstrated bright staining for CLC and MBP in 95% +/- 3% of cells in Eo-type colonies but only in 5% +/- 4% of cells in GM-type colonies. Radioimmunoassay for MBP was positive in 5/9 Eo-type and 0/10 neutrophil-macrophage (“GM-type”) colonies, with a mean level (nanogram/colony) of 11.6 +/- 4.2 per Eo-type colony; four of the latter colonies were doubly positive for both histamine and MBP. These and previous findings point out the morphological and biochemical heterogeneity of peripheral blood Eo-type colonies and provide direct evidence for the existence of a common, circulating basophil-eosinophil progenitor.

Expression of the gene encoding human eosinophil lysophospholipase, the Charcot-Leyden crystal (CLC) protein, was studied in transiently transfected COS cells. Recombinant CLC (rCLC) protein expression was demonstrated both by Western blot and radioimmunoassay inhibition analyses of transfected COS cell extracts and by immunofluorescent staining and ultrastructural immunogold analyses of intact cells. The rCLC protein was immunochemically indistinguishable from native eosinophil-derived CLC protein, and each transfected COS cell expressed approximately 11 pg of rCLC protein as determined by radioimmunoassay and assessment of transfection efficiency. Immunofluorescent microscopy and ultrastructural immunogold analyses localized rCLC protein to the nucleus, cytoplasm, and plasma membrane of COS cells. Lysates from transfected COS cells producing CLC protein expressed significant lysophospholipase activity. Furthermore, rCLC protein expressed in COS cells spontaneously formed the distinctive intracytoplasmic and intranuclear hexagonal bipyramidal crystals characteristic of the native eosinophil and basophil-derived protein. Expression of the CLC gene confirms the authenticity of the CLC cDNA, the expression of lysophospholipase activity by this unique eosinophil and basophil constituent, and will facilitate the routine purification of the active enzyme for in vitro and animal model studies of its role (or roles) in eosinophil and basophil associated allergic inflammation and eosinophil-parasite interactions.

We examined the ultrastructural localization of (a) a secondary granule matrix protein -eosinophil peroxidase (EPO) -by cytochemistry, (b) a secondary granule core protein (major basic protein, MBP) by immunogold labeling, and (c) a primary granule protein (the Charcot-Leyden crystal protein, CLC protein) by immunogold labeling in eosinophilic myelocytes (EMS) and mature, activated eosinophils that differentiated from umbilical cord blood progenitors cultured in the presence of recombinant human interleukin-5 (rhK-5). These studies provide the first substructural localization of MBP to condensing cores of immature secondary granules of EMS, as well as identification of unicompartmental, MBFrich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules mroduction Mature human eosinophils are polymorphonuclear granulocytes with two large granule populations (1). One of these populations consists of primary granules (2,3), which comprise ~5 % of the cytoplasmic large granules, are unicompartmental, increase in activated eosinophils in vivo and in vitro, and are the granule storage organelle for the Charcot-Leyden crystal (CLC) protein (2). The second large granule population consists of secondary (or specific) granules, which comprise ~9 5 % of the large cytoplasmic granules, are bicompartmental, undergo quantitative decreases in number and qualitative morphological changes in activated eosinophils in vivo and in vitro, and are the storage organelle for a number of eosinophil products (reviewed in 1). These include major basic protein (MBP) (4,5), eosinophil-derived neurotoxin (EDN) (4). eosinophil cationic protein (ECP) (4,5), eosinophil peroxidase (EPO) (1,5), and tumor necrosis factor-a ( m a ) (6). MBP is confined to the

In 1956 Gansler (1) observed infiltration of the rat uterus by eosinophils coincident with the estrus cycle. This observation was confirmed and extended by the work of Bassett (2) and Ross and Klebanoff (3). Since then, various investigations have shown (a) that injection of estrogen into castrated or immature rodents leads to immediate uterine eosinophilia (4-10), (b) that the uterine eosinophil is marrow derived (11), (c) that the uterine eosinophil number varies >100-fold during the normal estrus cycle (12), (d) that uterine content of eosinophil peroxidase varies directly with the estrus cycle (13,14), (e) that eosinophils possess a unique cell surface estrogen receptor (4-9), and (f) that estrogen-induced uterine eosinophilia apparently has no dependence upon uterine mast cell activity (15-18).In contrast, there are few observations relating eosinophils to human reproductive physiology . Cyclic eosinopenia correlating with ovulation has been reported (19-21) and cyclic variations in endometrial eosinophils and their uptake of tritiated estradiol have also been observed (22). However, no role for the eosinophil in normal human reproduction is presently recognized . During the course of studies of hypersensitivity diseases in pregnancy we found that serum levels of a molecule immunochemically similar to the eosinophil granule major basic protein (MBP)' were elevated in all pregnant women, increasing during pregnancy and decreasing to normal levels after parturition. Because eosinophilia is not a feature of normal pregnancy, and serum levels of other eosinophil proteins are not elevated, the results of this study suggest that the immunoreactive MBP in pregnancy serum is derived from a source other than the eosinophil . Materials and MethodsDithiothreitol, iodoacetamide, equine liver catalase, sodium borate, boric acid, Staphylococcus aureus protein A, chloramine T, protamine sulfate, bovine serum albumin, Trizma