THE RELATIONSHIP OF THE CHEMOTACTIC BEHAVIOR OF THE COMPLEMENT-DERIVED FACTORS, C3a, C5a, AND C567, AND A BACTERIAL CHEMOTACTIC FACTOR TO THEIR ABILITY TO ACTIVATE THE PROESTERASE 1 OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES

The inhibition profiles obtained when a series of p-nitrophenyl ethyl alkylphosphonates and of p-nitrophenyl ethyl chloroalkylphosphonates were used to interfere with the chemotactic activity of polymorphonuclear leukocytes stimulated by C3a, C5a, and bacterial factor were the same as found previously when C567 was the chemotactic agent. This indicates that as in the chemotactic activity induced by C567, an obligatory step in the chemotaxis caused by C3a, C5a, and bacterial factor is the activation of proesterase 1 of the rabbit polymorphonuclear leukocyte. C5a and C3a activate proesterase 1 of peripheral blood polymophonuclear leukocytes as measured by the increase of acetyl DL-phenylalanine β-naphthyl esterase activity. Attempts to detect in a like manner the proesterase 1 of the same leukocytes using bacterial factor under varying circumstances have consistently failed. It is concluded that bacterial factor, for unknown reasons, is unable to activate proesterase 1 to the same extent as the complement-derived chemotactic factors. The hypothesis of there being a quantitative difference in the ability of bacterial factor to activate proesterase 1 compared with the complement-derived factors explains the previous observations that bacterial factor can not deactivate to itself or to the complement-derived factors, although these latter factors can deactivate to themselves, to each other, and to the bacterial factor. The quantitative difference in the ability of bacterial factor to activate proesterase 1 compared to the complement-derived factors is also associated with and explains the finding that the maximal chemotactic activity attainable when bacterial factor is the chemotactic agent is distinctly less than that obtained using either C3a, C5a, or C567. These results indicate that the activation of proesterase 1 is a general requirement for the chemotactic activity of rabbit polymorphonuclear leukocytes with known macromolecular chemotactic agents and suggest that under several different circumstances the level of chemotactic activity attained is related to the degree of such activation

PARTIAL BIOCHEMICAL CHARACTERIZATION OF THE ACTIVATED ESTERASE REQUIRED IN THE COMPLEMENT-DEPENDENT CHEMOTAXIS OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES

It was shown in the preceding paper that incubation of the rabbit polymorphonuclearleukocyteswith phosphonate esters leads to an irreversible inhibition of the ability of the leukocyte to respond to the chemotactic factor. This "cell-dependent inhibition" was attributed to the inactivation by the phosphonates of an esterase existing in or on the leukocyte in an already activated state. As shown in this paper, incubating the leukocyte with phosphonate in the presence of certain esters prevents this cell-dependent inhibition. The protection is specific; the ester must be an acetate. Ethyl formate, ethyl propionate, ethyl butyrate, glucose 6-phosphate, fructose 1,6-diphosphate, ATP, tosyl arginine methyl ester, or acetyl tyrosine ethyl ester do not protect. The protection is independent of the phosphonate used to inhibit, and the degree of protection depends on the relative concentrations of acetate and phosphonate. Those acetates which protect are also the esters which inhibit chemotaxis when added to the leukocyte in the upper part of the chemotaxis chamber. It is concluded that the activated esterase is an enzyme capable of specifically splitting, or binding acetates, or doing both. Presumably the esterase is some type of acetylesterase or acetylase. The known aliesterase present in the leukocyte is not the activated esterase. Inhibition of the activated esterase by phosphonates has no effect on endogenous or exogenous glycolysis

MECHANISMS OF IMMUNOLOGIC INJURY OF RAT PERITONEAL MAST CELLS : II. COMPLEMENT REQUIREMENT AND PHOSPHONATE ESTER INHIBITION OF RELEASE OF HISTAMINE BY RABBIT ANTI-RAT GAMMA GLOBULIN

There is an absolute requirement for C'1, C'2, C'4, C'3, and C'5 in releasing histamine from rat peritoneal mast cells sensitized with rabbit anti-rat gamma globulin. This conclusion is based upon the restoration of histamine-releasing capacity by adding highly purified complement components to sera deficient in one or more of these components. Of special advantage was the availability of sera from humans with inborn or acquired deficiencies in a single component. The p-nitrophenyl ethyl phosphonates block this reaction by inhibiting an antigen-antibody-activated esterase which exists in a phosphonate resistant precursor state until activated by the interaction of the sensitized mast cell and serum complement. There is almost complete disparity between the ability of the phosphonates to inhibit complement-dependent histamine release by rabbit anti-RGG and to inactivate C'1a. Even though C'1a is required for complement-dependent histamine release by rabbit anti-RGG, this is not the esterase being blocked by the phosphonates under the experimental conditions used. The pattern of inhibition by the phosphonates of the antigen-antibody-activated esterase required for complement-dependent, noncytotoxic histamine release is remarkably similar to that of the esterase required for homocytotropic antibody-mediated histamine release. One possible implication is that these two quite different modes of carrying out antigen-antibody-induced histamine release from rat peritoneal mast cells lead to activation of the same esterase and share a common pathway

MECHANISMS OF IMMUNOLOGIC INJURY OF RAT PERITONEAL MAST CELLS : I. THE EFFECT OF PHOSPHONATE INHIBITORS ON THE HOMOCYTOTROPIC ANTIBODY-MEDIATED HISTAMINE RELEASE AND THE FIRST COMPONENT OF RAT COMPLEMENT

The ability of a number of p-nitrophenylethyl, alkyl phenylalkyl, chloroalkyl, and aminoalkyl phosphonates to inhibit the homocytotropic antibody-mediated release of histamine from rat peritoneal mast cells has been tested. The effectiveness of these same phosphonates against the activated first component of rat complement (C'1a) has also been investigated. The rat mast cell esterase activated by the reaction of antigen and homocytotropic antibody resembles chymotrypsin in its reactivity with the phenylalkyl and chloroalkyl phosphonate, but is unlike this protease in its greater responsiveness to the 5-aminopentyl phosphonate relative to the pentyl phosphonate. The antigen-homocytotropic antibody-activated mast cell esterase and chymotrypsin, thus, appear to be similar, but different enzymes; i.e., they are parazymes (see reference 4, p. 501). There are distinct differences in the pattern of inhibition given by the phenylalkyl and aminoalkyl and alkyl phosphonates of the homocytotropic antibody-mediated histamine release from rat peritoneal mast cells and from guinea pig lung slices. On the basis of these differences it is concluded that the esterases activated by the combination of antigen and homocytotropic antibody on the mast cells of the two species are not the same. The arithmetic dose response curve found for the action of the phosphonates on the antigen-induced histamine release from rat peritoneal mast cells contrasted sharply with the logarithmic relationship found when these same inhibitors acted on the guinea pig lung system. This suggests that in addition to the antigen-antibody-activated esterases being unlike, the detailed mode of histamine release from the mast cells of the guinea pig lung differs from that of the mast cells of the rat peritoneum. Distinct and large differences were found in the pattern of inhibition of histamine release from rat peritoneal mast cells and of rat C'1a given by the phenylalkyl, and chloroalkyl and alkyl phosphonates implying that esterase activated by the combination of antigen with the sensitized rat peritoneal mast cells is not C'1a. Thus, the results with the peritoneal mast cells lead to the same conclusion as the previous work with guinea pig lung slices; i.e., the antigen-antibody-activated esterase involved in the homocytotropic antibody-mediated release of histamine is not part of the complement system

RABBIT ANAPHYLACTIC ANTIBODY

Antibody capable of sensitizing rabbit skin for passive cutaneous anaphylaxis is produced in the rabbit as early as 6 to 7 days following antigenic stimulation. It reaches peak activity around the 9th day and is gone by the 3rd wk. The antibody is heat labile, sensitive to treatment with mercaptoethanol, non-precipitating and does not fix complement. In order to demonstrate PCA activity a latent period is required of from 48 to 72 hr after introduction of the antibody into the rabbit's skin; the activity can persist for at least 17 days. It has a faster electrophoretic mobility than rabbit γG-globulin, and is eluted somewhat earlier than γG-globulin from Sephadex G-200, although distinctly after γM-globulin. No relationship was demonstrated between the rabbit PCA activity and the hemagglutinating activity found in the same sera. The rabbit anaphylactic antibody differs in almost all properties studies from the rabbit 7S antibody capable of sensitizing guinea pigs for PCA which arises at the same time. This latter antibody found early in immunization had properties which were indistinguishable from those described for the rabbit 7S antibody giving PCA in the guinea pig found in late hyperimmune sera

PEPSIN DIGESTION OF RABBIT AND SHEEP ANTIBODIES : THE EFFECT ON COMPLEMENT FIXATION

7S sheep antibody is similar to 7S rabbit antibody, insofar as it too can be digested with pepsin to yield a 5S fragment still capable of precipitating homologous antigen. The 5S fragment from rabbit as well as sheep antibody is capable of fixing guinea pig complement. However, this fixation differs from the fixation by 7S antibody, since preformed antibody-antigen aggregates containing either sheep or rabbit 5S antibody fix only a maximum of 20 to 40 per cent of the total complement. The portion of complement which is fixable by the 5S preformed aggregates seems to be different from the remainder of the complement. Prior treatment of guinea pig serum with 5S washed preformed aggregates removes that portion of complement fixable by 5S antibody, and will leave the complement remaining unable to be fixed by further addition of 5S immune aggregates. Such absorbed complement, however, is as capable as is unabsorbed complement of being fixed by 7S antibody. The 5S sheep and rabbit antibody fixes the portion of complement which it is capable of fixing, as well as or better than the corresponding 7S antibody fixes the total complement. 5S preformed aggregates are broken down by treatment with cysteine, and their complement-fixing ability is lost

MECHANISMS OF THE INHIBITION OF CHEMOTAXIS BY PHOSPHONATE ESTERS

Studies in the time course of the response of rabbit polymorphonuclear leukocytes (PMN's) to the complement-associated chemotactic factor have revealed that the response is virtually complete by 60 min with less than 15% additionally responding cells thereafter. Phosphonate esters with a well defined capacity to inhibit serine esterases have been used to study the cell-associated enzymes of the rabbit PMN required for the chemotactic response. Two types of inhibition of the cell response to the chemotactic factor have been found: (a) cell-dependent inhibition occurring as a result of pretreatment of PMN's with phosphonate esters; and (b) chemotactic factor-dependent inhibition demonstrated only when the phosphonate ester is present during the chemotactic response. Differences were found in these two modes of inhibition caused by various phosphonates, in terms of their time course of inhibition, in the dose response curves, and in the structure-activity relationships. It has been conclusively demonstrated that the phosphonate esters have no direct inhibitory effect on the chemotactic factor. This has been shown by retention of activity of the chemotactic factor following incubation with phosphonate esters and subsequent removal by dialysis. In addition, the activity of the chemotactic factor and its physical-chemical characteristics in density gradient ultracentrifugation were unaltered in the continued presence of a potent phosphonate inhibitor of chemotaxis. The uptake of the dye trypan blue was studied in cells treated with phosphonate in such a manner to induce cell-dependent inhibition of chemotaxis. Even when 84% cell-dependent inhibition of chemotaxis occurred, no uptake of the dye by leukocytes was found. Thus, phosphonate-induced inhibition of cell responsiveness in chemotaxis was not associated with generalized cell damage as defined by exclusion of the dye. It is concluded that cell-dependent inhibition is due to the presence of a cell-bound esterase which is already activated and thus susceptible to inhibition by phosphonate esters before contact of the cell with the chemotactic factor. The second type of inhibition, chemotactic factor-dependent inhibition, is considered due to a cell-bound esterase which becomes susceptible to inhibition by phosphonate esters only after contact of the PMN with the chemotactic factor. It is postulated that the chemotactic factor activates this phosphonate-resistant precursor making it susceptible to the inhibitory action of the phosphonate ester

ESTERASES OF THE POLYMORPHONUCLEAR LEUKOCYTE CAPABLE OF HYDROLYZING ACETYL DL-PHENYL-ALANINE β-NAPHTHYL ESTER : RELATIONSHIP TO THE ACTIVATABLE ESTERASE OF CHEMOTAXIS

Previous published work has led to the hypothesis that the activatable esterase of chemotaxis is a serine esterase of the rabbit polymorphonuclear leukocyte existing in an inert, phosphonate insusceptible form, which after activation is capable of hydrolyzing aromatic amino acid esters and being inhibited by phosphonates. In the present study, directed to the testing of this hypothesis, we have shown that rabbit peritoneal polymorphonuclear leukocytes contain three esterases capable of hydrolyzing the aromatic amino acid ester, acetyl DL-phenylalanine β-naphthyl ester. Two of these esterases, esterase 1 and esterase 2, are inhibited by various p-nitrophenyl ethyl phosphonate esters. The inhibition of each esterase is irreversible and progressive with time. When the logarithm of the esterase activity remaining after cell and inhibitor have been in contact for a constant time is plotted against the concentration of inhibitor, a straight line results. These results support the conclusion that both esterases are serine esterases. The third esterase, esterase 3, differs from the other two by its inability to be inactivated by any of the phosphonates no matter how high the concentration of phosphonate or prolonged the period of incubation of cell with phosphonate. The activity of esterase 1 is at least 10,000 times more easily inhibited by phosphonates than is that of esterase 2; incubating rabbit polymorphonuclear leukocytes for 15 min at 27°C with 10–9–10–8 M concentrations of various phosphonates inactivates esterase 1, but it required 10–6–10–4 M concentrations of the same phosphonates to inhibit esterase 2. The inhibition profiles of esterase 1 are distinctly different from those of esterase 2 when the two esterases are tested with the phenylalkylphosphonates, chloroalkylphosphonates, and alkylphosphonates. The inhibition profile of esterase 1 is essentially the same as that of the activatable esterase of chemotaxis obtained previously when the same three homologous series of phosphonates were tested for their ability to protect against deactivation by the chemotactic factor or give chemotactic-dependent inhibition. It is tentatively concluded that esterase 1 of the rabbit peritoneal neutrophil is the activated form of the activatable esterase of chemotaxis

A COMPARISON OF THE SPECIFICITY OF INHIBITION BY PHOSPHONATE ESTERS OF THE FIRST COMPONENT OF COMPLEMENT AND THE ANTIGEN-INDUCED RELEASE OF HISTAMINE FROM GUINEA PIG LUNG

The ability of a number p-nitrophenylethyl alkyl, phenyl alkyl, chloroalkyl, and aminoalkyl phosphonates to inhibit the activated first component (C'1a) of guinea pig complement, and the antigen-induced release of histamine from sliced, perfused guinea pig lung has been compared. C'1a in its reactivity with these phosphonates is distinctly more similar to trypsin than to any of the other enzymes studied previously. It is suggested that both trypsin and C'1a possess an anionic group in the active center of the respective enzyme, but the distance between the anionic and esteratic site in C'1a might be less than in trypsin. The pattern of inhibition of histamine relase by the alkyl, phenyl alkyl, and chloroalkyl phosphonates is similar to the inhibition of C'1a by these compounds, although distinct differences are apparent. The aminoalkyl phosphonates are distinctly less active inhibitors of histamine release than the corresponding alkyl phosphonates, whereas the reverse is true of the inhibition of C'1a. On the basis of these differences, it is tentatively concluded that the organophosphorus-inhibitable enzymes in the guinea pig systems studied here are similar but not identical

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