FUNCTION OF THE RETICULOENDOTHELIAL SYSTEM : I. A STUDY ON THE PHENOMENON OF CARBON CLEARANCE INHIBITION

The inhibitory effect of heat-aggregated albumin on carbon clearance has been studied with respect to the dose-dependent relationships involved. It has been found that the dose of aggregated albumin does not affect the degree of inhibition but does affect the duration of inhibition. Above a critical dose the duration increases exponentially with increasing albumin dose. The addition of gelatin to the carbon suspension was found to produce two effects: (a) it prolonged the inhibitory effect of a given aggregated albumin dose, and (b) it produced a slowing in the rate of carbon removal. The fact that foreign red cells inhibit carbon clearance in a manner similar to denatured albumin suggests that the relationships observed relative to denatured albumin may be generally applicable to carbon clearance and indeed may have application to phagocytic particles other than carbon.     

THE ROLE OF OPSONINS IN THE CLEARANCE OF LIVING AND INERT PARTICLES BY CELLS OF THE RETICULOENDOTHELIAL SYSTEM

An investigation of the clearance of bacteria and colloids from the blood stream of mice has shown that both living and inert particles require serum factors (opsonins) in order that they may be phagocytosed by the macrophages of the reticuloendothelial system. It has been demonstrated that after the injection of a large dose of colloid there is a depletion of these serum opsonins which appears to account for the reduced rate of clearance of a second dose of colloid or living bacteria, since replacement of these factors leads to normal clearance. The significance of these results is discussed and it is suggested that in "blockaded" animals there is a depletion of serum opsonins rather than a saturation of phagocytic cells.     

FURTHER OBSERVATIONS ON THE MECHANISM OF RETICULOENDOTHELIAL BLOCKADE

The mechanism of the reticuloendothelial "blockade" which followed injection of large quantities of chromic phosphate without exogenous stabilizing material was investigated in Wistar rats. The RE blockade observed for several hours after induction appeared related to the continuing circulation of the chromic phosphate-blockading dose, and a reduction in the size of the particles used enhanced blockade. RE blockade appeared to be particles specific and was not related to a generalized depression of RE-phagocytic cell function. Studies in isolated perfused rat livers appeared to eliminate saturation of particle-specific macrophage clones as a likely explanation of blockade, and blockade could not be explained on the basis of depletion of serum opsonins. In the system employed, it is postulated that blockade occurs when large numbers of circulating particles saturate specific macrophage cell membrane-binding sites rather than from physical stuffing of RE-phagocytic cells.     

STUDIES OF THE HUMAN LYMPHOCYTE RECEPTOR FOR HEAT-AGGREGATED OR ANTIGEN-COMPLEXED IMMUNOGLOBULIN

The lymphocyte receptor for complexed immunoglobulin was shown not to bind heat-aggregated human serum albumin, bovine serum albumin, transferrin, F(ab')₂, reduced and alkylated Ig, and mildly oxidized Ig, which indicated that the receptor is specific for a site dependent on disulfide bond(s) on the Fc portion of complexed Ig. Inhibition experiments provided evidence that the same receptor binds both heat-aggregated Ig and antigen-antibody complexes. Lymphocytes treated with pronase were no longer able to bind Ig complexes, which suggested that the receptor is a protein or glycoprotein. Additional evidence was obtained that lymphocyte surface Ig and the receptor for complexed Ig are distinct since the former could be capped without affecting the distribution of the latter, and surface Ig was not detectable after trypsinization of lymphocytes, whereas the binding of Ig complexes was unaffected by such treatment. Incubation of lymphocytes which had bound Ig complexes in tissue culture medium at 37°C revealed that the complexes remained on the surface membrane for several hours, and that only a minority of lymphocytes binding complexes showed cap formation. Lymphocytes which had heat-aggregated IgG specifically bound to their receptors for complexed Ig were markedly inhibited in their ability to mediate antibody-dependent cytotoxicity, thus providing strong evidence for the necessity of the receptor in this immune activity. Titration of this inhibition with varying amounts of complexes revealed distinct plateaus in the dose-response curve. This suggested that there may be more than one kind of receptor and/or different populations of lymphocytes which bear the receptor.     

THE ROLE OF THE RETICULOENDOTHELIAL SYSTEM IN MOUSE ANAPHYLAXIS AS TESTED WITH HOMOLOGOUS ANTIBODY-ANTIGEN COMPLEXES

Blockade of the RES was accomplished by the intravenous injection of carbon particles, thorotrast, zymosan, or a suspension of Bordetella pertussis. The blockade resulted in a decrease in sensitivity to anaphylaxis produced by the intravenous injection of soluble antigen-antibody complexes consisting of an optimal shocking mixture of bovine plasma albumin and mouse antibody to this antigen. The decrease in sensitivity to anaphylaxis was dependent on the dose of blockading agent and on the time between blockade and challenge with complex. The loss of sensitivity to anaphylaxis could not be restored by the administration of fresh serum from normal mice nor by guinea pig complement. Antigen-antibody complexes were rapidly removed from the blood with an average half-time of 11.9 minutes in normal mice. Complexes were cleared at significantly more rapid rates in mice previously sensitized to antigen. Although not all the results can be explained on the basis of blockade the facts indicate that the RES does play an important but as yet undefined role in passive homologous anaphylaxis in the mouse.     

THE DISTRIBUTION OF THE IMMUNE BODIES OCCURRING IN ANTIPNEUMOCOCCUS SERUM

The immune bodies of antipneumococcus serum are completely precipitated by 38 to 42 per cent. saturation with ammonium sulphate. They are incompletely precipitated by (a) ammonium sulphate in less than 38 per cent. saturation, (b) saturation with sodium chloride, (c) dilution and saturation with carbon dioxide, (d) removal of crystalloids by dialysis. The immune bodies of antipneumococcus serum are, therefore, associated or combined with that fraction of the globulins precipitated by 38 to 42 per cent. saturation with ammonium sulphate. The immune body fraction does not correspond exactly with the ordinary euglobulin (one-third saturation with ammonium sulphate or complete saturation with sodium chloride) or with the insoluble globulins precipitated by carbon dioxide or dialysis. These fractions carry with them only a part of the immune bodies. Neither the albumin nor that fraction of the globulin not precipitated by 38 to 42 per cent. saturation of ammonium sulphate contain any of the demonstrable antibodies. The most promising method for the practical purification of the immune bodies occurring in antipneumococcus serum appears to be precipitation by 38 to 42 per cent. saturation with ammonium sulphate.     

DELAYED HYPERSENSITIVITY : I. EFFECT OF IN VITRO EXPOSURE OF CELLS TO ANTIGEN UPON LEUKOCYTIC TRANSFER OF DELAYED HYPERSENSITIVITY

Exposure to picryl guinea pig albumin with 3–6 picryl groups per mole failed to affect the ability of peritoneal exudate or peripheral blood leukocytes from sensitized donors to transfer delayed sensitivity to normal recipients. In contrast, conjugates containing 40–48 picryl groups per mole altered the ability of exposed leukocytes to transfer delayed sensitivity. Evidence is presented that highly conjugated guinea pig albumin is self-aggregating. Lightly conjugated albumin, previously heat-aggregated, also was effective in "desensitization." The properties of antigen size, cell association of antigen after exposure, and desensitization appear to be associated.     

MECHANISMS OF ENDOTOXIN TOLERANCE : I. RELATIONSHIP BETWEEN TOLERANCE AND RETICULOENDOTHELIAL SYSTEM PHAGOCYTIC ACTIVITY IN THE RABBIT

Pyrogenic tolerance following 7 daily intravenous injections of 2.0 µg/kg E. coli endotoxin in albino rabbits was associated with significant increases in RES phagocytic activity as measured with colloidal carbon. Nevertheless, 4 hours after RES blockade with thorotrast (3 ml/kg), the tolerant rabbits exhibited significantly lower fever indices following intravenous endotoxin challenge than did non-tolerant control animals despite comparably depressed capacities to clear carbon from the blood. Moreover, plasma from rabbits tolerant to endotoxin induced significant tolerance in normal rabbits prepared by thorotrast blockade without enhancing the depressed carbon clearance. This passive protection extended to heterologous endotoxins. Analysis of the data indicates that RES blockade does not abolish tolerance; rather blockade resets the reactivity to endotoxin in the normal and tolerant animal, rendering both exquisitely reactive, but permitting retention of the major portion of tolerance. Apparently the tolerant animal possesses a dual endotoxin defense system. One system is abolished by thorotrast; the other is in part humoral, accounts for the greater portion of tolerance, and is thorotrast-resistant. The nature of the humoral component is not defined but is consistent with that of an opsonin with high endotoxin specificity.     

THE DEACTIVATION OF RABBIT NEUTROPHILS BY CHEMOTACTIC FACTOR AND THE NATURE OF THE ACTIVATABLE ESTERASE

As shown previously, immune complexes engender in rabbit serum a factor capable of inducing chemotaxis of rabbit polymorphonuclear leukocytes. This chemotactic factor consists of a complex of the fifth, sixth, and seventh components of complement. As demonstrated here, the polymorphonuclear leukocytes incubated with such treated rabbit serum lose their ability to respond chemotactically to the chemotactic factor. They are "deactivated." The process of "deactivation" is a function of the duration of contact of the cells with, and the concentration of, the treated serum. There is a parallelism between the time course of deactivation and of chemotaxis, as well as the dose-response curves for the two processes. Chemotactic factor purified by isoelectric precipitation and ion-exchange chromatography produces deactivation in the same manner as the treated serum. The deactivating activity requires, as does the chemotactic factor, the sixth component of complement; like the chemotactic factor, it is heat-stable and nondialyzable. Deactivation is prevented by the same phosphonate esters shown previously to prevent chemotaxis by the complement-associated chemotactic factor. The profiles of the phosphonates in protecting against deactivation are the same as the profiles for the chemotactic factor-dependent inhibition of chemotaxis. Aromatic amino acid derivatives prevent both chemotaxis and deactivation. We conclude from this evidence that the chemotactic factor is able to deactivate or induce chemotaxis depending upon experimental conditions. The fact that the profiles given by the phosphonates for protection against chemotactic factor-dependent deactivation and for chemotactic factor-dependent inhibition of chemotaxis are the same indicates that the "activatable esterase" is involved in both processes. Acetate esters such as ethyl acetate and others shown previously to prevent chemotaxis by inhibiting the "activated esterase" do not prevent deactivation. This indicates that deactivation can occur without participation of the latter enzyme, implying that deactivation involves only a part of the biochemical mechanism of chemotaxis. The protection against deactivation afforded by aromatic amino acid derivatives is specific, insofar as nonaromatic amino compounds and simple acetate esters have no effect. In addition, as stated, the aromatic amino acid derivatives inhibit deactivation and chemotaxis by the chemotactic factor. This latter finding, together with the demonstration of the involvement of the activatable esterase in both deactivation and chemotaxis, suggests that the activatable esterase of the rabbit polymorphonuclear leukocyte is a serine esterase with a special affinity for aromatic amino acid derivatives.     

THE MECHANISM OF BLOCKADE OF THE RETICULOENDOTHELIAL SYSTEM

Rats were injected intravenously with various particulate materials with and without the addition of various stabilizing agents. One hour after the injection of the blockading colloid, a tracer dose of similar or different colloidal material was injected. A half-time clearance rate of the tracer dose greater than 4 minutes was taken as evidence of blockade of the RES. Blockade of the tracer dose occurred when the surface properties of the particles in both blockading and tracer doses were identical. Different particles stabilized by the same agent behaved as identical particles and identical particles stabilized by different agents behaved as different particles. The opsonization of a tracer dose of gelatin-stabilized colloid in vitro by a specific globulin fraction obtained from heterologous and homologous plasma prevented its blockade, while opsonization of both blockading and tracer doses with the same proteins resulted in blockade. However, when both blockading and tracer doses were opsonized with isologous globulins, blockade did not occur.     

Absence of a binding reactivity of human C-reactive protein for immunoglobulin or immune complexes

Because C-reactive protein (CRP) has been identified as a component of circulating immune complexes from patients with inflammatory diseases, we sought to evaluate a potentially clinically important interaction of this acute-phase protein with immunoglobulin or experimentally-prepared immune complexes in vitro. Highly purified human CRP was incubated with a variety of immunoglobulin substrates, including monomeric immunoglobulin G1 (IgG1), a polyclonal IgG, heat-aggregated IgG, and human serum albumin/anti-serum albumin complexes. We were unable to detect a significant binding interaction of radioiodinated CRP with any of these materials, using either polyethylene glycol (PEG) precipitation or sucrose density gradient ultracentrifugation. In contrast, binding of radioiodinated human C1q to both aggregated immunoglobulin and immune complexes was readily detected by these techniques. Incubation of radiolabeled CRP with serum samples from 22 patients with active inflammatory diseases and high levels of circulating immune complexes disclosed no difference in the amount of PEG-precipitable CRP when compared with serum samples from healthy individuals. However, a radiolabeled commercial preparation of CRP did result in some PEG-precipitable radioactivity after incubation with aggregated IgG. These findings provide no support for a biologically important binding interaction of CRP with immunoglobulin or immune complexes, and they suggest that highly purified preparations of CRP should be used in functional studies of this acute-phase protein.     

THE DYNAMICS OF RETICULOENDOTHELIAL BLOCKADE

The dynamics of "reticuloendothelial blockade" were studied in living rabbits and isolated, perfused rabbit livers utilizing gelatin as a blockading agent and Au¹⁹⁸ stabilized in gelatin as a tracer. Employing the above experimental model, the following observations were made. (a) RES blockade was specific and dependent on the surface properties of the particle under study. (b) RES blockade was not caused by saturation of hepatic removal mechanisms. (c) RES blockade was not caused by depletion of demonstrable serum opsonins. (d) RES blockade appeared to correlate with high circulating levels of the blockading agent, per se. Thus, under the conditions employed, the term "reticuloendothelial blockade" was a misnomer. Although specificity of liver macrophage-particle interaction was evident and deserves further study, the data suggest that blockade as usually studied is a laboratory phenomenon induced by the continuing circulation of the blockading agent.     

THE IGA SYSTEM : I. STUDIES OF THE TRANSPORT AND IMMUNOCHEMISTRY OF IGA IN THE SALIVA

1. Five patients with congenital or acquired agammaglobulinemia, lacking detectable IgA in serum or saliva, were transfused with 1 to 2 liters of normal plasma. In 2 of these patients IgA was demonstrated in parotid saliva collected after transfusion, but in none of the 5 was salivary IgG or IgM found. This observation indicates the selective transport of IgA into saliva. 2. The observation by others of an immunochemical difference between serum and sahvary IgA globulin was confirmed. In contrast to serum IgA, salivary IgA is attached to a protein having antigenicity which migrates as a gamma₁ globulin. We have termed this protein component "transport piece". 3. The transport piece has been found in an unbound form in the saliva of persons completely lacking IgA: agammaglobulinemic patients, ataxia-telangiectasia patients, a healthy person lacking IgA, and a newborn infant. Free transport piece still occurs in the normal child's saliva after IgA production begins. By adulthood there is usually no free transport piece in the saliva. 4. Heat-aggregated salivary IgA, like heat-aggregated serum IgA, does not fix complement. 5. Our findings offer support for the view that there is a distinct local antibody system for the protection of the mucous surfaces.     

STUDY OF THE ADSORPTION ON AND DESORPTION FROM POLYSTYRENE-HUMAN SERUM ALBUMIN CONJUGATES OF RABBIT ANTI-HUMAN SERUM ALBUMIN ANTIBODIES HAVING DIFFERENT SPECIFICITIES

An insoluble specific adsorbent for anti-human serum albumin antibodies was prepared by coupling human serum albumin (H.S.A.) to polystyrene by azo bonds. Rabbit anti-H.S.A. immune serum was passed through a column of the adsorbent. It was shown that different volumes of the immune serum were required for the saturation of the different determinant groups of H.S.A. by their corresponding antibodies. The elution of the anti-H.S.A. antibodies adsorbed on the column was achieved by passing successively through the column an acetate buffer pH 3.0 and a solution of 0.1 N HCl in 0.15 M NaCl. The antibodies were eluted in three different fractions, each of which was composed of γ-globulins only. These three fractions contained different proportions of antibodies of different specificities.     

Facilitation of penicillin haptenation to serum proteins.

Traditionally, penicillin binding to serum proteins was believed to be a passive chemical process; however, it appears to be facilitated by serum factors. The objectives of this in vitro investigation were to examine facilitated penicillin haptenation, to study the kinetics of haptenation, and to determine the nature of haptenation-facilitating factors. The model involved addition of [3H]benzylpenicillin to serum or albumin solutions (at pH 7.3 to 7.4) and incubation at 37 degrees C for up to 72 h. The extent of penicillin binding to proteins in serum was found to be four- to fivefold higher than with solutions having comparable concentrations of purified albumin, total protein, or total immunoglobulin. Ultrafiltration of serum reduced penicillin binding to serum proteins substantially. An ultrafiltrable haptenation-facilitating factor(s) was found to be less than 0.5 kDa but was not calcium or magnesium. Finally, the extent of penicillin binding was related to albumin purity, as binding substantially increased with albumin purity. These findings suggest that there is a factor(s) in serum that facilitates covalent binding of penicillin to serum proteins. The factor(s) can be removed and then restored to increase penicillin binding to albumin. It appears that at least one component of the facilitation factor is less than 0.5 kDa, which suggests that it is not a peptide and that it is some simple serum component other than calcium or magnesium.     

STUDIES ON THE DIMENSIONS OF THE RABBIT ANTI-BENZYLPENICILLOYL ANTIBODY-COMBINING SITES

Rabbit antisera prepared against conjugates of the benzylpenicilloyl (BPO) bifunctional haptenic group were analyzed to determine whether the antibodies are adapted to only a portion of the large BPO molecule, or to the entire molecule, and whether specificity extends to the lysine side chain and adjoining structures of the immunizing carrier protein. No antibodies adapted to the phenylacetylamine portion of the BPO group could be detected in a pooled rabbit anti-BPO serum globulin fraction by PCA and quantitative precipitin analysis using several phenylacetylamine-protein conjugates as antigens. No antibodies adapted only to the thiazolidine carboxylic acid portion of the BPO molecule were detected in the anti-BPO globulin fraction using quantitative precipitin and hapten inhibition methods. At least the bulk of the anti-BPO antibodies was found to be adapted to the entire BPO haptenic group. By quantitative hapten inhibition of precipitation of the anti-BPO globulin fraction, the anti-BPO antibodies were found to show specificity for a 6 carbon amide side chain corresponding to the lysine side chain through which BPO groups are bound predominantly to protein. The contribution of this 6 carbon chain to antibody-hapten binding was small; (–ΔF°) was calculated to be 460 calories per mole (average). Rabbit anti-BPO antibodies prepared against BPO-rabbit serum albumin conjugates showed specificity also toward structures of the immunizing carrier protein, and possibly toward secondary or tertiary structural configurations. Penicilloyl conjugates of rabbit serum albumin precipitated from 3 individual rabbit antisera more anti-BPO antibodies than did penicilloyl conjugates of heterologous carriers (poly-L-lysine, human serum albumin, and human γ-globulin). Anti-BPO antibodies demonstrated heterogeneity with regard to closeness of fit to the haptenic group, or with regard to the dimensions of the combining sites, or both. It was concluded that at least a large part of anti-BPO antibodies are specifically adapted to a large antigenic unit comprised of the entire BPO group, the lysine side chain, and structural configurations of the immunizing carrier protein.     

Structural changes in human serum albumin induced by ingestion of acetylsalicylic acid

Acetylsalicylic acid (aspirin) acetylates human serum albumin under physiologic conditions in vitro. These investigations were done to determine whether this phenomenon occurs in vivo and to delineate the site(s) of acetylation on the albumin molecule.Albumin was reacted in vitro with aspirin labeled with (14)carbon at the acetyl-1 or the carboxyl carbon. The altered albumin was hydrolyzed with trypsin and peptide mapping performed. Albumin so treated contains a unique peptide, designated "A," and shows diminution of two normal peptides, designated "B" and "C." Peptide "A" is never seen in normal albumin. Amino acid analyses indicate that peptide "A" equals the sum of peptides "B" and "C." Furthermore all three peptides contain lysine but lack arginine. Thus peptide "A" is formed by the acetylation of a lysine residue which is normally susceptible to trypsin and yields peptides "B" and "C." Radioautography of the peptide maps show most of the acetyl-1-(14)C activity in peptide "A." This indicates that one of the lysine residues in this peptide is the preferential site for the transacetylation reaction.Peptide "A," used as a marker for acetylation, is found in albumin from patients who take aspirin but is not demonstrable in albumin from one of these patients while she was taking sodium salicylate. A transacetylation reaction between aspirin and human albumin occurs in vivo and is similar to that observed in vitro.     

ELECTROPHORETIC STUDIES ON ELEMENTARY BODIES OF VACCINIA

Electrophoretic studies were made on vaccine virus, collodion particles, and glass particles suspended in 0.01 molar buffer solutions at pH 7.9, in which the moving boundary method was used. In some experiments, uncoated particles were used; in others, particles were coated with proteins and then resuspended in the buffer solution after a washing; in still others, an excess of protein which had been used to coat the particles was included in the buffer medium. Streaming boundaries were obtained with all dilute suspensions of particles in solutions containing no soluble protein instead of the flat ones usually observed with the Tiselius moving boundary technique. This boundary artifact was suppressed by maintaining a density gradient of sufficient magnitude in association with the moving boundary to counteract the tendency of endosmotic flow. This was done partially by increasing the concentration of the particles in the suspensions, and almost completely by retaining an excess of soluble-coating substance in the solutions containing the particles. The mobility of elementary bodies of vaccinia corresponds to that found for the heat-stable (S) antigen. This value was not altered by drying, heating, ether extraction, or simple washing, but was materially increased by treatment with the surface active detergent (duponol) which presumably altered the nature of the surface of the virus particles. Collodion particles coated with the heat-stable antigen of vaccinia had the same mobility as elementary bodies under comparable conditions. Glass particles coated with normal rabbit serum moved at the rate of albumin, the fastest serum component in the buffer solutions used. However, both collodion particles and vaccine virus moved at a somewhat slower rate when they were similarly coated and measured in the presence of an excess of serum in the solutions. This was probably due to adsorption of a small amount of one of the slower components (globulin) of rabbit serum on the surface of the particles. Simple washing after treatment seemed to remove the coating of serum proteins, at least in part, from both collodion particles and elementary bodies of vaccinia.     

STUDIES ON BACTERIEMIA : V. THE EFFECT OF SIMULTANEOUS LEUKOPENIA AND RETICULOENDOTHELIAL BLOCKADE ON THE EARLY BLOOD STREAM CLEARANCE OF STAPHYLOCOCCI AND ESCHERICHIA COLI

Observations are presented on the course of experimental bacteriemia in rabbits modified by thorotrast reticuloendothelial "blockade" and mechlorethamine induced leukopenia, given singly and together. The production of severe granulocytopenia did not impair the clearance of staphylococci from the blood stream and did not enhance the impairment of clearance produced by thorotrast reticuloendothelial "blockade." Neither severe granulocytopenia nor thorotrast blockade alone produced consistent impairment in the removal of circulating E. coli from the blood stream, whereas when these conditions were present together, the early phases of clearance in these animals were virtually prevented. These results indicate that the host mechanisms operative in the control of bacteriemia differ for staphylococci and E. coli. It is suggested that the fate of bacteria within polymorphonuclear leukocytes may in part determine the importance of these cells in the removal of bacteria from the blood stream.     

THE OCCURRENCE DURING ACUTE INFECTIONS OF A PROTEIN NOT NORMALLY PRESENT IN THE BLOOD : II. ISOLATION AND PROPERTIES OF THE REACTIVE PROTEIN

Methods are described for isolating a protein commonly present in the blood of patients during the acute phase of various infections which, unlike the normal serum proteins, is precipitable by the C polysaccharide of Pneumococcus. The reactive protein is present in the fraction of serum albumin precipitated by either ammonium or sodium sulfate between 50 and 75 per cent saturation. From this fraction the reactive protein separates out on dialysis against tap water. Following removal of the alcohol-ether-soluble lipids from acute phase serum the reactive protein becomes soluble in tap water, and is no longer precipitable by traces of calcium but still retains its precipitability with the C polysaccharide.