The mediation of tissue eosinophilia in hypersensitivity reactions. II. Separation of a delayed eosinophil chemotactic factor from macrophage chemotactic factors.

In anaphylactic cutaneous lesions induced by DNP-ascaris extract in the guinea-pig, the time-course of delayed tissue eosinophilia was found to parallel that of the macrophage reaction, reaching its peak in 24 h. Macrophages could be differentiated from lymphocytes by the numerous lysosomal granules which stained for acid phosphatase. Extracts from such skin lesions contained a delayed eosinophil chemotactic factor and two different macrophage chemotactic factors. Most of the delayed eosinophil chemotactic factor was separated from the two macrophage chemotactic factors by gel filtration on Sephadex G-100 and Sephadex G-200 in that order. The eosinophil chemotactic factor after re-chromatography on Sephadex G-I99 showed no or little chemotactic activity for macrophages.     

Preparation of antisera to the μ chain of IgM

The protein fraction in the first peak obtained by Sephadex G-200 gel filtration of normal bovine serum was reduced with 2-mercaptoethanol, alkylated with iodoacetamide and recycled on a Sephadex G-200 column. The resultant chromatogram indicated the presence of two peaks the second of which was found to be pure monomeric IgM. Immunization of rabbits with the second peak protein resulted in the production of an antiserum with both heavy and light-chain activity. The light-chain activity was simply removed by absorption of the antiserum with glutaraldehyde-polymerized material obtained from the second peak of the initial Sephadex G-200 eluate, yielding an antiserum monospecific for the heavy chain of the IgM molecule.     

Lymphocyte Chemotaxis in Inflammation. IV. Isolation of Lymphocyte Chemotactic factors from PPD-induced Delayed Hypersensitivity Skin Reaction Site in the Guinea-pig. with Special Reference to a Factor Chemotactic for B Cells

Lymphocyte chemotactic factors were extracted from PPD-induced delayed hypersensitivity skin reaction sites in guinea-pigs. The presence of 3 chemotactic factors (LCF-a, LCF-b and LCF-c) was suggested by gel filtration on Sephadex G100. LCF-a was purified by gel filtration on Sephadex G-15 followed by peptide mapping. This substance was considered to be a thermostable and dialysable peptide; it appeared to be effective for B cells but not for T cells.     

The Chemotactic Activity of Normal and Complement-Activated Serum as Measured by the Leading-Front Method Using a Boyden Chamber

The chemotactic activity in serum, defined as the attractant effect of serum on the migration of neutrophil granulocytes (PMN) has been investigated for the purpose of characterizing the major chemotactic factors in serum as measured by the leading-front technique, using a modified Boyden chamber. The chemotactic activity was measured in fresh and heated normal and activated serum and in serum fractions thereof separated by gel filtration. By gel filtration on Sephacryl S-200 a partly heat-labile C3-C5-associated chemotactic factor with molecular weight between 70,000 and 150,000 was isolated from fresh normal serum. The heat-labile chemotactic activity was destroyed by pronounced complement activation. Gel filtration of complement-activated serum on a Sephacryl S-200 column showed the existence of one C5-associated chemotactic factor with approximately 70,000 molecular weight and one unidentified factor with approximately 150,000 molecular weight, whereas no low molecular weight chemotactic activity was demonstrated. On the other hand, gel filtration of activated serum on a Sephadex G-75 column demonstrated one C5-associated chemotactic factor of approximately 70,000 molecular weight and one 10,000-50,000 molecular weight factor active only in the presence of 2% normal serum. This investigation suggests that the chemotactic activity in fresh normal serum is mediated by a partly heat-labile C3-C5-associated complex. In activated serum three chemotactic factors were demonstrated, one unidentified factor with 150,000 mol wt and two C5-dependent factors with 70,000 and 10,000-50,000 mol wt, the latter probably corresponding to C5a desarg. Accordingly, this study also suggests that C5a is not the only chemotactic factor generated in serum.     

Production of chemotactic factor for lymphocyte by digestion of IgG with a highly purified polymorphonuclear leukocyte neutral thiol proteinase

In the present study, we purified a neutral thiol proteinase from dog PMN leukocytes and indicated that the proteinase elaborated the chemotactic factor for lymphocytes by cleavage of IgG. The neutral thiol proteinase was purified about 744-fold by ion-exchange chromatographies and affinity chromatography, and the final preparation was over 70% pure. After incubation of dog IgG with the proteinase, three distinct protein peaks were seen by the gel filtration on Sephadex G-200. Only the third peak, perhaps a dialyzable peptide, showed a significant chemotactic activity for dog lymphocytes.     

Studies on Alternaria allergens. I. Isolation of allergens from Alternaria tenuis and Alternaria solani.

Extracts of Alternaria tenuis and Alternaria solani were separated into dialyzable (molecular weight less than 10,000) and non-dialyzable forms. The latter was further fractionated by gel filtration through Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The dialyzable material was fractionated by gel filtration through Sephadex G-50. The allergenic activities of the fractions obtained from the A. tenuis extract was measured in vitro by the radioallergosorbent test assay and the allergenic potency was measured by radioallergosorbent test inhibition assay. Allergenic activity was detected in most of the non-dialyzable fractions, the majority of the activity being in the last G-100 fraction (MW approximately 20,000) which was predominantly protein in nature. The same component may be responsible for the activity found in the dialyzate and its first G-50 fraction since the immunodiffusion studies indicated that the last G-100 fraction has antigenic components in common with those of the first G-50 fraction. In addition, cross-reactions between A. tenuis and A. solani extracts show that the two species share common antigenic determinants.     

Novel neutrophil chemotactic factor derived from human peripheral blood mononuclear leucocytes.

Human mononuclear leucocytes isolated from the peripheral blood by centrifugation on Ficoll-Hypaque cushions and adherent on plastic petri dishes, produced a chemotactic factor that attracted human neutrophilic granulocytes to the same extent as did optimal concentrations of the complement split product C5a and the leukotriene B4. The active component eluted from a Sephadex G-50 gel filtration column as a single peak with an apparent molecular weight of 10,000. The chemotactic activity was resistant to reductive cleavage of disulfide bonds and heating at 100 degrees C for 30 min but was lost when reduction and heating were combined. Digestion with a proteolytic enzyme eliminated the attractive potential. The data suggest that this is a novel chemotactic peptide. It is conceivable that it has been seen previously and was mistaken for a lymphokine or interleukin 1.     

Further characterization and biologic activity of ragweed antigen--induced neutrophil chemotactic activity in man.

We have previously described the appearance in serum of increased neutrophil chemotactic activity (NCA) during bronchospasm induced by inhalation of ragweed antigen in ragweed-sensitive subjects. This NCA is non-complement derived, appears within 1 min after antigen inhalation, and is not seen after methacholine-induced bronchospasm. This article describes further characterization of this chemotactic activity and correlation with in vivo leukocytosis. NCA consistently eluted in the void volume (fraction I) after Sephadex G-150 chromatography of patient serum obtained 10 min postchallenge. Fraction I contained 94% of the NCA of postchallenge whole serum. Both postchallenge whole serum and fraction I deactivated neutrophils to autologous chemoattractants and complement-derived chemotactic factors, but not serum-independent chemotactic factors. NCA was chemotactic for neither human nor guinea pig eosinophils, nor for human mononuclear cells. A significant increase of circulating neutrophils was seen only after antigen-induced bronchospasm and correlated with the increase in NCA. Thus, NCA represents another inflammatory mediator of probable mast cell origin that may explain, at least partially, the accumulation of neutrophils observed in the peripheral blood, skin, and bronchial wall after immediate hypersensitivity reactions.     

Correlation between mature and immature thymocytes. Immature thymocyte potentiating factor (IMPF) produced by mature thymocytes.

Low-dense mature thymocytes (MT) and high-dense immature thymocytes (IMT) were investigated. Immature thymocyte-potentiating factor (IMPF) was found in the supernatant from MT stimulated by Con A. On the other hand, suppressing activity was present in the supernatant from the unstimulated IMT. IMPF was different from Con A itself. Sephadex G-100 gel chromatography of IMPF revealed that the activity was recovered at the position between bovine serum albumin and ovalbumin.     

Inability to block the activity of guinea pig migration inhibitory factor with di-isopropyl phosphorofluoridate

Studies were carried out to confirm a previous report that the activity of migration inhibitory factor (MIF) was blocked by di-isopropyl phosphorofluoridate (DFP), suggesting it acted as a protease. MIF was obtained by the stimulation of guinea pig lymphocytes with concanavalin A and the culture supernatants were chromatographed on Sephadex G-100 gel columns. The MIF-rich fraction was incubated with DFP or with other organophosphorus esterase inhibitors; the inhibitors were removed by dialysis, and the media were tested for MIF activity. The results, reported here, indicate that guinea pig MIF activity is not destroyed by treatment with serine esterase inhibitors.     

Further purification and characterization of heat-stable enterotoxin produced by Yersinia enterocolitica.

Heat-stable enterotoxin (ST) of Yersinia enterocolitica was produced under defined conditions. It was first detected in the culture supernatant of the late-logarithmic phase of growth and increased lineally during the the stationary phase of growth. The ST level became maximum at the decline phase of growth, and the ST was not detected in the lysate of bacteria obtained from the decline phase of growth. The ST was extensively purified from the culture supernatant, and about a 1,905-fold purification was achieved with a yield of 8.9%. The minimal effective dose of the purified ST was approximately 25 ng in the suckling mouse assay. The purified ST gave a single 280-nm absorbing peak on polyacrylamide disc gel electrophoresis and had a maximum absorption at 272 nm, and its molecular weight was 9,700 by Sephadex G-75 superfine gel filtration. The biological activity of the purified ST was lost by treatment with 2-mercaptoethanol, suggesting that the ST contained disulfide bridges in the molecule which were required for the development of toxic activity. The purified ST was heat stable at 100 degrees C for 10 min between pH 2.2 and 8.0, but not at pH values greater than 9.0 or in 2 N HCl. The treatment of the ST with trypsin resulted in a retarded elution of the ST activity by Sephadex F-75 superfine gel filtration and a passage through a UM-20 membrane filter.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2. Copper ions significantly increased enzyme activity, while Zn⁺⁺ and Mn⁺⁺ caused marked inhibition.     

Purification of adenovirus type 12 tumor antigen from transformed hamster cells

The adenovirus type 12 (Ad12) tumor (T) antigen has been isolated and purified from Ad12-transformed baby hamster kidney cells. The immunoreactive material that precipitates from the 105,000 g supernatant of cell extract at 35% saturation of ammonium sulfate has been characterized by a number of column procedures: gel filtration on Sephadex G-100, hydroxylapatite chromatography, DEAE cellulose chromatography, and affinity chromatography on single-stranded and double-stranded DNA cellulose. The native immunoreactive material appears to have a size of 80,000 daltons on Sephadex G-100, elutes as a single peak in hydroxylapatite and DEAE cellulose, does not bind to single-stranded DNA, but has a strong affinity for double-stranded DNA. A small portion (<15%) of complement-fixing material elutes from double-stranded DNA at pH 6.2 in 0.4 M NaCl, while the rest of the activity elutes at pH 8.0. Although each of these procedures achieves considerable purification of T antigen, the resulting preparations contain multiple peptide species as detected by SDS-polyacrylamide gel electrophoresis. Different combinations of these column procedures achieve 1000-fold purification of T antigen, and resulting preparations contain four to seven peptide species on SDS-polyacrylamide gel electrophoresis. The relative quantities of individual peptides and identity of some of them vary among preparations obtained by different purification procedures.     

Lymphocyte chemotaxis in inflammation. IX. Further characterization of lymphocyte chemotactic lymphokines produced by purified protein derivative-stimulation in vitro and in vivo

As recently reported, one lymphocyte chemotactic factor (beta-LCF, mol. wt. about 27,000) released from activated guinea-pig lymphocytes appeared to be identical to one of the LCFs (LCF-d) isolated from extract of purified protein derivative (PPD)-induced delayed-type hypersensitivity skin reaction sites in guinea-pigs with respect to antigenicity and chemotactic effect for T cells. However, the mol. wt. of LCF-d (about 300,000) was clearly distinct from beta-LCF. The experiments were undertaken to clarify the problem. beta-LCF appeared to be bound to some protein of normal guinea-pig serum (GPS) because the chemotactic activity was revealed in the fraction corresponding to that of LCF-d when the mixtures of beta-LCF with GPS were applied to a Sephadex G-200 column. Additionally, binding experiments using fluorescein isothiocyanate (FITC)-labelled beta-LCF were performed; fluorescence was only detected in the chemotactic fraction. It was thus assumed that the lymphokine (beta-LCF) would be released from activated lymphocytes around the inflammatory tissue, then bound with serum protein exuded in the site and function as LCF-d. The possibility was supported by the evidence that beta-LCF like-chemotactic substance (mol. wt. about 27,000) was dissociated from LCF-d under acid conditions. The factor dissociated from LCF-d was also bound with GPS protein under neutral conditions and converted to high molecular substance resembling LCF-d physiochemically. Furthermore, the chemotactic activity of LCF-d was almost completely absorbed by antibody against GPS. It is thus considered that the chemotactic activity of LCF-d may be attributed to beta-LCF released from activated lymphocytes and that some serum protein which binds beta-LCF may function as a carrier protein in the DTH sites.     

Depression of grass pollen-induced lymphocyte transformation by serum from hyposensitized patients.

Serum from grass-sensitive patients who had received hyposensitization therapy was found able to depress the in vitro response to Holcus lanatus grass pollen extract of lymphocytes from untreated grass-sensitive individuals. The inhibitory activity could no longer be demonstrated at serum dilutions equal to or greater than 1/100 and was not seen when the cells were stimulated with PHA or unrelated antigens. It was contained in the precipitated fraction of 33% saturated ammonium sulphate-treated serum, segregated with the 7S peak of Sephadex G-200 and with the IgG fraction of DEAE-Sephadex-fractionated serum and could be removed by the absorption of the serum with the Holcus lanatus allergenic extract. These data suggest that the serum factor responsible for the depressed in vitro lymphocyte response to the allergen in hyposensitized patients was a 7S IgG antibody induced by immunotherapy.     

The natural mediator for PMN emigration in inflammation: I. Purification and characterization of leucoegresin from Arthus skin site

A chemotactic factor (leucoegresin) for polymorphonuclear leucocytes (PMN) of rabbit, guinea-pig, rat and mice was extracted in the pseudoglobulin fraction of Arthus skin lesions and then highly purified by chromatography using Sephadex G-50, DEAE-Sephadex A-50 and CM-Sephadex C-50 in this order. Chemotactic activity was estimated by Boyden's method during the process of purification. Leucoegresin behaved as a homogeneous substance on ultracentrifugation and on moving boundary electrophoresis. This factor was a protein free of nucleic acid and its molecular weight was approximately 140,000 when measured by gel filtration. The sedimentation coefficient of this substance was 6.58S and its isoelectric point was pH 5.0. It was relatively heat-stable.

Leucoegresin induced pronounced PMN migration, but did not increase vascular permeability; PMN emigration occurred only at the site of venules.

Leucoegresin satisfied such criteria for the natural mediator of Arthus leucotaxis as local availability, parallel between activity (amount) and time-course of the reaction, and histological resemblance to the Arthus reaction.

     

Further characterization of the antigens of canine prostatic fluid

Fractionation of the precipitating components of canine prostatic fluid was carried out by gel filtration on Sephadex G-100. By gel filtration three fractions were obtained which were referred to as A, S, and F. Fractions S and F were further purified by recycling through a column of Sephadex G-100. The partially purified components, which were designated S₁ and F₂, appeared antigenically similar on the basis of gel diffusion precipitation experiments. Further characterization of these components was carried out in order to explain the asymmetric shortening of the precipitin line of fraction S₁ and the complete dissolution of the line of fraction F₂ when high concentrations of fraction F₂ were used. Quantitative precipitation studies, with the fractions and an anti-canine prostatic fluid serum, showed an unusually narrow zone of equivalence for fraction F₂. Fraction S₁, on the other hand, gave a much broader equivalence region. The R values for fractions S₁ and F₂ were found to be 8.5 and 15.0, respectively. Proteolytic determinations showed the presence of caseinolytic activities in both fractions S₁ and F₂; the activities per mg of these fractions were of the same magnitude. In view of the available immunological data it is suggested that the dissolution of the precipitin lines is due to the creation of a zone of antigen excess provided by high concentrations of fraction F₂.     

The chemical mediation of delayed hypersensitivity skin reactions. IV. Activation of chemotactic factor precursor by a trypsin-like protease in guinea pig plasma.

A single-chain precursor protein for macrophage chemotactic factor was previously reported (Ueda et al, Am J Pathol, 1982, 108:291-298). Apparent chemotactic activity was spontaneously generated in the precursor fraction during a long period of incubation, correlating with the cleavage of the precursor molecule into a two-chain protein. Generation and the limited proteolysis were both inhibited by phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, suggesting the presence and the role of a serine protease in the fraction. A serine protease was actually activated and separated from the precursor fraction with a benzamidine-conjugated cellulose affinity column. The protease was heat-labile, and the substantial molecular weight was 20,000 by gel filtration on a Sephadex G-75 column and 23,000 by SDS-polyacrylamide gel electrophoresis. It hydrolyzed [3H-acetyl casein and also fluorogenic synthetic substrates, butyloxycarbonylphenylalanylserylarginine methylcoumarylamide and butyloxycarbonylisoleucylglutamylglycylarginine methylcuomarylamide, and was inhibited by diisopropylfluorophosphate, PMSF, trasylol, soybean trypsin inhibitor, and tosyllysine chloromethylketone, but not by tosylphenylalanine chloromethylketone, chymostatin, p-chloromercuricbenzoate or ethylenediaminetetraacetate. By incubation of the precursor with the protease, rapid generation of chemotactic activity for macrophages was observed. The fact that the chemotactic factor newly generated in vitro was identical with a macrophage chemotactic factor previously separated from extract of delayed type hypersensitivity skin sites immunologically and physicochemically suggested an essential role of the protease in macrophage chemotactic factor generation in the delayed hypersensitivity skin reaction.     

Demonstration of a pancreatic proelastase 2-alpha 1-protease inhibitor complex in normal human plasma

A peak of immunoreactive pancreatic elastase 2 with a molecular weight consistent with that of a complex of elastase 2 and alpha 1-protease inhibitor (also referred to as alpha 1-antitrypsin) can be detected by radioimmunoassay in normal human serum or plasma (Geokas et al., J. Biol. Chem. 252:61-67, 1977). This material has been purified by gel filtration on Sephadex G-200 and by ion-exchange chromatography on DEAE-cellulose. The alpha 1-protease inhibitor-bound immunoreactive elastase 2 has been dissociated by incubation with hydroxylamine, and the resulting immunoreactive product isolated by gel filtration on Sephadex G-100. The dissociated immunoreactive elastase 2 was shown by affinity chromatography on turkey egg white inhibitor-bound agarose, before and after activation by bovine trypsin, to consist only of proelastase 2. A second peak of immunoreactive material associated with the high molecular weight fraction of plasma has been shown to result from a specific interaction of the 125I-labeled phenylmethanesulfonyl-elastase 2 employed as tracer in the radioimmunoassay with alpha 2-macroglobulin, resulting in apparent immunoreactivity. These results demonstrate that all of the detectable immunoreactive pancreatic elastase 2 in normal human plasma is proelastase 2 bound to alpha 1-protease inhibitor.     

Studies on the chemical composition and biological properties of transfer factor

Dialyzable transfer factor (dTF) was fractionated on Sephadex G-10 and G-25 fine columns, and biological activity was found in 3 fractions. One of these, designated VIa, and having a tendency to adsorb to the Sephadex G-10 gel, was shown to have a therapeutic effect on certain immunological diseases. Analysis of this fraction on thin-layer and gas chromatography and with infrared and mass spectroscopy indicated that about half of this fraction was composed of uracil; additional unidentified heterocyclic and aromatic substances were present in this fraction. Adjacent fraction V contained tyrosine and a small polyribonucleotide, and fraction VII hypoxanthine and additional unidentified components. Our results suggest that the therapeutic activity of dTF is not mediated through an immunologically specific informational molecule, but is rather based on non-specific stimulation of the expression of the immune response.