The binding of human lactoferrin to mouse peritoneal cells.

Human iron-saturated Lf (FeLf), which was labeled with 125I or 50Fe, was found to combine with the membrane of mouse peritoneal cells (MPC) which consisted of 70% macrophages. The following experimental data suggested the involvement of a specific receptor. (a) The binding of FeLf to MPC reached a saturation point. (b) The binding of radioactive FeLf was inhibited by preincubating the cells with cold FeLf but not with human Tf, human aggregated and nonaggregated IgG, or beef heart cytochrome c (c) Succinylation and carbamylation of FeLf resulted in a loss of its inhibiting activity on the binding of radioactive FeLf. Removal of neuraminic acid from FeLf increased its inhibitory activity. (d) The ability of apoLf to inhibit the binding of FeLf to MPC was significantly lower than that of FeLf. The existence of a Lf receptor capable of concentrating Lf released from neutrophils on the membrane of macrophages could explain the apparent blockade of the release of iron from the reticuloendothelial system, which accounts for the hyposideremia of inflammation. A receptor for FeLf was also found on mouse peritoneal lymphocytes. The affinity constant of FeLf for both lymphocytes and macrophages was 0.9 X 12(6) liter/mol. Howerver, macrophages bound three times more FeLf molecules (20 X 10(6)) per cell than did lymphocytes (7 X 10(6)).

Incidence and specificities of IgA and IgM anti-AgG autoantibodies in various mouse strains and colonies.

Mice, greater than 20 wk old, were tested for the presence of anti-IgG autoantibodies by agglutination and radioimmunoassay. IgA and IgM anti-IgG were found in the 129/Sv, C57BL/6, and DBA/2 strains from the local colony at the International Institute of Cellular and Molecular Pathology (ICP), at the Institut Pasteur de Paris (IP), and in the endotoxin-resistant C3H/He strain of ICP. These strains were negative at Iffa Credo (IC), and at The Jackson Laboratory (JL). Among 33 strains from the latter colony, 129/J, AKR/J, CBA/J, C57L/J, and NZB/BinJ were positive. All were specific pathogen-free and, excepting the NZB/BinJ, are not known to develop systemic autoimmune disorders. These differences between colonies suggest an influence of the environment on the production of anti-IgG. Evidence for the role of an infectious agent was provided by the fact that germ-free DBA/2 were negative in contrast to their SPF relatives. Strains which were positive at ICP and IP for anti-IgG had four-times higher serum levels of total IgA and two-times higher levels of total IgG than the corresponding negative strains from IC and JL. The anti-IgG titers differed markedly from one strain to the other in the same environment; e.g., in mice from ICP, BALB/c mice produced 40-times less anti-IgG than 129/Sv. IgA anti-IgG occurred only in high producers of anti-IgG. In these animals, the proportion of IgA vs. IgM anti-IgG was very different from one group to the other; C57BL/6 had mainly IgM anti-IgG, DBA/2 mainly IgA anti-IgG, and 129/Sv both IgM and IgA anti-IgG. The IgA anti-IgG from 129/Sv, 129/J, NZB/BinJ, C57L/J, DBA/2, and C3H/He had restricted hetero-, iso-, and allotypic specificities. It reacted only with mouse IgGa2, but not with the Ig-1b allotype. C57BL/6 also had IgA anti-IgG with a narrow specificity, but directed against IgG1 without allotypic restriction. In contrast to the specificity of IgA anti-IgG, the antibody activity of IgM anti-IgG was much broader, except in the 129/Sv and 129/J strains where IgM anti-IgG shared the same narrow specificity with IgA.

Age-dependent production of IgA and IgM autoantibodies against IgG2a in a colony of 129/Sv mice.

Although much of the basic immunological work has been done with mice, little is known about anti-IgG autoantibodies in this species. Dresser (1, 2) has reported the occurrence, in CBA mice, of anti-IgG antibody (Ab)(1) detected by a hemolytic-plaque assay after stimulation with endotoxin or immunization against sheep erythrocytes. IgM rheumatoid factor has also been described in various strains of mice with a systemic lupus erythematosus-like disease (3). Recently, we have tried to induce anti-IgG in mice of the 129/Sv strain by inoculating autologous IgG. To our surprise, we found that the sera of all the animals had, before any inoculation, anti-IgG detectable by agglutination of particles coated with autologous IgG. The possibilities to investigate the mechanism of production and the biological role of this kind of Ab prompted us to undertake a study of the nature and specificity of the mouse anti-IgG.

The effect of complement on the ingestion of soluble antigen-antibody complexes and IgM aggregates by mouse peritoneal macrophages.

Complement was found to stimulate markedly the ingestion of soluble antigen-antibody complexes by mouse peritoneal macrophages. This was shown indirectly by measuring the release of degradation products when the complexes were labeled with 125I, or directly when the antigen, that was human transferrin, was labeled with 59Fe. In this case, the metal which was released from human transferrin inside the cells was not excreted, and its accumulation in the macrophages was a direct index of the uptake of immune complexes. The decay of radioactivity in macrophages after ingestion of 125I-labeled complexes was similar when they were taken up with or without complement, indicating that complement acts primarily on ingestion and not on digestion or excretion. The ingestion of complexes was morphologically confirmed using fluorescein-labeled antigen in the immune complexes. The opsonic effect of complement was also observed with IgM aggregates indicating that soluble complexes can be ingested through complement receptors without involvement of Fc-receptors, as required for particulate antigen-antibody complexes.

Lactoferrin, an iron-binding protein in neutrophilic leukocytes.

Lactoferrin, an iron-binding protein previously shown to occur in many external secretions, is identified as one of the major proteins present in human and guinea pig neutrophilic polymorphonuclear leukocytes. The identification of this protein in leukocyte extracts was based upon a comparison of its electrophoretic, antigenic, and iron-combining properties with the corresponding properties of the same protein isolated from human and guinea pig milk. Immunochemical quantitations showed that lactoferrin occurs in human neutrophilic leukocytes at the concentration of 3 microg per 10(6) cells. Tissue cultures from guinea pig bone marrow and spleen actively synthesized the protein, as shown both by net production of lactoferrin and incorporation of labeled amino acids into the protein. Immunohistochemical data indicate that lactoferrin first appears in myeloid cells at the stage of the promyelocyte.

The involvement of lactoferrin in the hyposideremia of acute inflammation.

The hyposideremia of inflammation was found to be based on a three-step mechanism involving lactoferrin, the iron-binding protein from the specific granules of neutrophilic leukocytes. (a) Lactoferrin is Released from Neutrophils in an Iron-Free Form. When phagocytosis was induced in neutrophils by zymosan or bacteria, lactoferrin was recovered in the incubation medium together with other constituents of the specific granules, such as alkaline phosphatase and lysozyme. Lactoferrin extracted from leukocytes was able to bind the amount of iron corresponding to its theoretical iron-binding capacity. After injection of endotoxin into rats, lactoferrin was detected in various tissues where it was normally absent, or in the plasma when the reticuloendothelial system (RES) had previously been blocked by injections of India ink or aggregated albumin. (b) Lactoferrin is Able to Remove the Iron from Transferrin. Significant exchange of iron from transferrin to lactoferrin was observed in vitro only at a pH below 7.0 or in the presence of a high concentration of citrate. However, the fast elimination of lactoferrin in vivo, when saturated with iron, might account for the observed transfer of iron to endogenous or administered apolactoferrin. Intravenous injection of human apolactoferrin into rats caused a marked decrease of the plasma iron level. The kinetics of this process, as well as controls with other proteins, ruled out the possibility of a secondary inflammatory effect due to phlogogenic contaminants. (c) Fe-Lactoferrin is Taken-up by the RES. By immunofluorescence, lactoferrin was shown to be bound and ingested by monocytes. The rate of elimination of human Fe-lactoferrin injected into rats was particularly fast when compared to that of human apolactoferrin, succinylated Fe-lactoferrin, or other human proteins. Blockade of the RES slowed down the rate of clearance of Fe-lactoferrin and was also found to retard the elimination of endogenous rat lactoferrin released by endotoxin. These experiments suggest the existence of specific receptors for Fe-lactoferrin on the membrane of macrophages.

The ingestion and digestion of human lactoferrin by mouse peritoneal macrophages and the transfer of its iron into ferritin.

Human lactoferrin (Lf) labeled with 125I and/or 59Fe was found to be ingested in vitro by mouse peritoneal macrophages (MPM). The uptake measured after 15 h incubation reached a saturation point at a concentration of 200 microgram/ml in the culture medium, whatever was the iron content of Lf. In such conditions, the uptake of transferrin (Tf) used as a control was 10 times lower. At a concentration of 80 microgram/ml in the medium, one cell picked up about 0.7 X 10(6) molecules of Lf per hour, and 0.13 X 10(6) molecules of Tf per hour. Iron-saturated Lf disappeared from MPM with a half life of 14.5 h, whereas the halflife of iron-free Lf was 4.2 h. Concomitant with the intracellular digestion of Lf, the iron was transmitted to ferritin. These data provide additional support for the hypothesis that Lf plays a key role in iron turnover, especially at the level of the reticuloendothelial system where iron is recovered from the catabolism of erythrocytes.

Association of lactoferrin with specific granules in rabbit heterophil leukocytes.

Lactoferrin has been identified in rabbit heterophil leukocytes on the basis of its immunological reactivity, electrophoretic mobility, acid-resistant iron-binding properties, and spectral characteristics. Leukocyte lactoferrin was found to be exclusively localized in the specific (secondary) granules, which have been resolved from other subcellular components by zonal differential centrifugation and by isopycnic equilibration.

Studies on bovine cervical mucin. I. The amino acid composition and N-terminal amino acid of bovine oestrus cervical mucin.

Bovine oestrus cervical mucin, isolated by gel filtration on Sepharose 6B, was found to be homogeneous (i) in electrophoresis on cellulose acetate at pH 8.6 after release of sialic acid and in acrylamide-agarose gel at pH 8.7 in the presence of sodium dodecyl sulphate, where not a single compact zone was found after staining for proteins, (ii) by analytical ultracentrifugation and equilibrium centrifugation in a CsCl density gradient; as well as (iii) by immunoelectrophoresis, which revealed two kinds of antibodies against mucin, reacting, respectively, with the primary structure and with determinants depending on the integrity of the glycoprotein structure. Alanine was identified as the N-terminal amino acid of the peptide core, which appeared to consist of a repetition of a 32-35 residue sequence, viz. (Thr)8, (Glu, Pro, Ala, Val)3, (Asp, Leu)2 and (Arg, Cys, Ileu)1. No integral numbers could be assigned to (Ser)3.4, (Gly)2.3 and (Phe)0.6 which were also present.

Esterase activity associated with secretory IgA and free secretory component preparations from human milk.

Secretory immunoglobulin A (IgA) and free secretory component were purified from human milk using methods involving (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography. It was found that the preparations, although apparently pure by conventional criteria based on immunoelectrophoresis, zone electrophoresis, or ultracentrifugation, were consistently contaminated by two different esterases. These enzymes could apparently not be removed by further ion-exchange chromatography and/or gel filtration. They could, however, be eliminated by passage on an immunoabsorbent made from anti-free secretory component antiserum.

The recognition by monoclonal antibodies of various portions of a major antigenic site of human growth hormone.

The reactivities of five mouse monoclonal antibodies against human growth hormone (hGH) were defined by either a competitive radioimmunoassay with insolubilized antibodies or by an agglutination-inhibition method with hGH-coated polystyrene particles. The five antibodies reacted significantly but to various degrees with human placental lactogen and at least three antibodies reacted with human prolactin and three synthetic peptides extending from residues 19 to 128, 73 to 128 and 98 and 128 of hGH. Four tested monoclonal antibodies failed to react with bovine growth hormone and with hGH oxidized by performic acid. The antibodies were further distinguished by their different reactions with hGH modified by reduction and alkylation or by adsorption on a polystyrene surface. The unique specificity of each antibody was confirmed for most of them by an agglutination method in which the agglutinating activity of hGH was tested on latex particles coated with various paired combinations of the monoclonal antibodies. The lack of agglutination with certain combinations suggested that the specificities of such a pair of antibodies overlapped each other. These results suggest that the sequences corresponding to the synthetic peptides participate in the structure of a major antigenic site of which various portions are recognized by the monoclonal antibodies.

The same region of human growth hormone is involved in its binding to various receptors.

The Fab fragments of three monoclonal antihuman GH (anti-hGH) antibodies, among five tested, inhibited the binding of the hormone to the receptors of the human lymphoid cell line IM-9 and liver membranes of the pregnant rabbit. The results were similar for the receptors of human lymphocytes and rabbit liver, suggesting that both receptors reacted with the same region of the hormone. The Fab fragments of the most inhibitory antibody also inhibited the down-regulation by the hormone of the receptors on human lymphocytes. The fragments of this antibody completely blocked the binding of the hormone to the receptors of the rabbit liver, despite the fact that this carries two or more classes of receptors. Therefore, all of these various receptors apparently interact with the same region of the hormone. Three synthetic peptides extending from residues 19-128, 73-128, and 98-128 failed to inhibit the binding of hGH to its lymphocyte or liver receptors however, these peptides reacted significantly with the monoclonal antibody, which was the strongest inhibitor of the interaction of hGH with the cellular receptor, confirming that the receptor-binding site of the hormone is in the amino-terminal part of the molecule and suggesting indirectly that the short sequences 98-128 participates in the constitution of the receptor-binding site of the hormone.

Specificities of antibodies to human growth hormone (hGH) in patients treated with hGH: longitudinal study and comparison with the specificities of animal antisera.

The specificities of human and animal antibodies (Abs) against human GH (hGH) were analyzed using competition experiments with five monoclonal antibodies to hGH (MAbs). The results indicate that 1) the Abs produced by patients receiving long term hGH therapy as well as Abs of goat, rabbit, and mouse origin recognized the various hGH epitopes defined by the MAbs; 2) the proportion of each Ab population, with a given specificity, differed markedly in different patients and also with time in the same patient; 3) the titer of certain Ab populations was very low in some patients, and 4) polyclonal mouse Abs and some human Abs enhanced the binding of [125I] hGH to insolubilized MAbs.

Particle counting immunoassay (PACIA). III. Automated determination of circulating immune complexes by inhibition of an agglutinating factor of mouse serum.

A factor capable of agglutinating human IgG coated particles (latex) has been found in mouse serum. This factor (MAG) was used in an unpurified form to detect circulating immune complexes in the particle counting immunoassay (PACIA) system, which allows measurement of agglutination with great precision. MAG did not react with monomeric IgG, nor with reduced and alkylated aggregated IgG. It was inhibited by immune complexes in small antigen excess. Among the various subclasses of IgG, IgA and IgM, only IgG1 and IgM when coupled to Sepharose beads displayed an inhibitory activity towards MAG. That the inhibitory factors detected in serum were immune complexes or aggregated Ig was suggested by the correlation obtained with the amounts of 'heavy' IgG found in the serum samples by Ultrogel chromatography and by the polydisperse distribution of the inhibitory factors in the heavy fraction of gradient ultracentrifugation.

Inhibition of idiotype-anti-idiotype reaction in particle counting immunoassay as a tentative assay of IgE.

Rabbit antibodies were raised against a purified mouse monoclonal IgG1 antibody having specificity for the D epsilon 2 determinant of human IgE. After appropriate absorption this antiserum was anti-idiotype specific and was used to set up a particle counting immunoassay for human IgE. Latex particles coated with F(ab')2 fragments of the anti-idiotypic antibodies were agglutinated by the monoclonal anti-IgE antibody (20 ng/ml). This reaction was inhibited up to 100% by human IgE and the assay took 30 min to perform with a sensitivity of 40 IU/ml. The coefficient of correlation with a routine IgE assay was r = 0.96, and the mean analytical recovery tested on 10 sera was 95.8%.

Particle counting immunoassay (PACIA). II. Automated determination of circulating immune complexes by inhibition of the agglutinating activity of rheumatoid sera.

Antigen--antibody complexes were detected in patients' sera by inhibition of the agglutinating activity of rheumatoid sera toward IgG coated particles (latex). Precision, sensitivity (1--10 microgram/ml equivalents of heat-aggregated IgG), and reproducibility (maximum coefficient of variation of 11%) were obtained by measuring agglutination with an instrument counting the residual free particles. Automation allowed testing of 20 to 40 samples per hour. The inhibitory activity of spontaneously agglutinating sera was determined after inactivating endogenous rheumatoid factor by reduction with dithiothreitol. Non-aggregated IgG did not significantly interfere. The agglutinating activity of 6 rheumatoid sera was tested after incubation with the various immunoglobulin classes and subclasses polymerized by coupling to agarose, and all were found to be readily absorbed by IgG1, but poorly by IgG3, IgA1 and IgM. Reactivity with IgG2, IgG4 and IgA2 clearly differed for different rheumatoid sera. Among 70 sera from blood donors, 7 had abnormally high inhibitory activity. Six of these had also an abnormal protein profile, suggesting existence of latent disease. High inhibitory activity in 15 sera out of 18 from patients with systemic lupus erythematosus, and in 23 sera out of 46 from patients with breast cancer suggested that the rheumatoid factor inhibition test has a discriminatory capacity comparable with that of more sophisticated techniques requiring radioisotopes and/or cellular material.

Typing of subclasses and light chains of human monoclonal immunoglobulins by particle counting immunoassay (PACIA).

The subclasses of monoclonal IgGs and IgAs were identified by particle-counting immunoassay. The principle of the test is the inhibition of the agglutinating activity of either specific antisera or monoclonal antibodies (for IgA only) on latex particles coated with a monoclonal IgG or IgA of known subclass. The feasibility of assay of polyclonal Ig subclasses was demonstrated. However, the anti-IgG2 antiserum cross-reacted with an allotype (nG4m(b)) of IgG4. The possibility of typing monoclonal Igs for light chains by the same technique was also demonstrated. Results are obtained in 30 min, and the method requires only small amounts of purified immunoglobulins (Igs) and antisera or monoclonal antibodies.

Particle counting immunoassay. IV. The use of F(ab')2 fragments and N epsilon-chloroacetyl lysine N-carboxyanhydride for their coupling to polystyrene latex particles.

Improved performance with the latex agglutination immunoassay, PACIA, is possible when the F(ab')2 fragments of antibody are chemically coupled at their hinge region to a protein-coated latex using a new coupling reagent, N epsilon-chloroacetyl lysine N-carboxyanhydride. This reagent, whether by orientating the complete antibody molecule or its F(ab')2 fragment or by preventing antibody denaturation, increased the sensitivity of the PACIA method. The use of F(ab')2 fragments eliminated most serum interference with the test.

Particle counting immunoassay (PACIA). I. A general method for the determination of antibodies, antigens, and haptens.

By using a device designed for counting blood cells, it is possible to measure the agglutination of polystyrene beads (0.8 mu) with accuracy and great sensitivity, the agglutination resulting in a reduction in the number of particles. The latter coated with antigen can be used for determining IgM or IgG antibodies e.g. human rheumatoid factor or rabbit anti-bovine serum albumin antibodies. Macromolecules with multiple antigenic determinants agglutinate particles carrying specific antibodies. This system has been applied for determining HPL and alpha1-fetoprotein with a threshold of sensitivity of about 10 microgram/1. However the agglutination was decreased by serum factors which led to a 10-fold loss of sensitivity. The interference of rheumatoid factor which agglutinated the particles coated with rabbit or goat immunoglobulins could be avoided by reduction of the serum to be analyzed with 5mM dithiothreitol for 5 min. Haptens, i.e. DNP-lysine and T4, were determined by their inhibitory activities toward their specific antibodies, the agglutinator being a hapten-macromolecule conjugate or the antibodies themselves.

Particle counting assay for anti-toxoplasma IgG antibodies. Comparison with four automated commercial enzyme-linked immunoassays.

An assay for anti-toxoplasma IgG antibodies based on agglutination of latex particles was set up and compared with commercial immunoassays. The reaction was measured by instrumental counting of particles remaining unagglutinated. The running time was 45 min. This test (PaC) was compared using 243 serum samples with four automated commercial immunoassays: the Enzymum test Toxo IgG (ES300, Boehringer), the Vidas Toxo IgG (Biomérieux), the IMX Toxo IgG (Abbott), the Magia Toxoplasma gondii IgG (Merck). The mean values (+/- SD) obtained by IMX (25 IU +/- 68) and ES300 (45 IU +/- 142) were significantly lower than the values obtained by Vidas (73 IU +/- 237, p < 10(-4) and p = 0.006, respectively), by Magia (80 IU +/- 300, p < 10(-4) and p = 0.0005) and by PaC (70 IU +/- 260, p < 10(-4) and p = 0.0126). The correlations between PaC and Toxo IgG Boehringer, Biomérieux, Abbott, Merck were r = 0.97, r = 0.98, r = 0.94, r = 0.98, respectively. The correlation coefficients between the enzyme-immunoassays ranged from 0.96 to 0.99. All positive samples by PaC were found to be positive by enzyme-immunoassays except for eight sera which were doubtful positives by the Enzymum test ToxoIgG from Boehringer. No negative sample by PaC was found positive by any of the enzyme-immunoassays. In PaC, when two latex preparations coated with different antigen were compared, the correlation was rather weak (r = 0.93) suggesting that the selection of the antigen can be critical. In conclusion, the four automated commercial immunoassays now available gave similar results. However, the discrepancies observed in this study underlined the importance of clinical and biological follow-up of the patients and the necessity to confirm the result. The introduction of a new technique such as PaC, which is now available for a large variety of assays in Clinical Chemistry and Microbiology, is justified by its intrinsic advantage of homogeneity. Therefore, automation is easy as well as the control of possible interference.