BY J. L. MONGAR AND H. 0. SCHILD From the Department of Pharmacology, University College London

Transcription

1 272 J. Physiol. (I958) I40, THE EFFECT OF CALCIUM AND ph ON THE ANAPHYLACTIC REACTION BY J. L. MONGAR AND H. 0. SCHILD From the Department of Pharmacology, University College London (Received 16 September 1957) In two previous papers (Mongar & Schild, 1957 a, b) we have studied the effects of inhibitors of anaphylaxis and formulated a scheme for the anaphylactic reaction based on the action of these inhibitors. The present paper is a continuation of this work, and deals mainly with the effects of calcium and ph on the anaphylactic reaction. Several immunological reactions are known to require calcium. Levine, Cowan, Osler & Mayer (1953a, b) have shown that calcium is required for immune haemolysis and that it is concerned with the binding of complement to the antigen-antibody complex. Humphrey & Jacques (1955) found that calcium lack inhibits the release of 5-hydroxytryptamine from platelets, which occurs during the antigen-antibody reaction in rabbit serum. The release of histamine by antigen from the formed elements of rabbit's blood is inhibited by oxalate and citrate but high concentrations of calcium (20 mm) also produce inhibition (McIntire, Roth & Richards, 1949). Citrate also inhibits certain allergic skin reactions (Rocha e Silva, 1952). The present work shows that the release of histamine from sensitized guinea-pig lung in anaphylaxis is inhibited by calcium lack and by a reduction in ph and that the effects of calcium and ph on the anaphylactic reaction are interdependent. A brief report of these findings has already been published (Mongar & Schild, 1957c). METHODS Guinea-pigs were actively sensitized with egg albumin and samples of chopped lung were prepared by the method previously described (Mongar & Schild, 1956) with the following minor modifications: For the study of the effects of ions the chopped lung was washed in isotonic sucrose instead of Tyrode solution. For the study of the effect of ph, phosphate or bicarbonate buffer was used, and in each case the final ph of the solution after incubation with the tissue was determined electrometrically. One millilitre of isotonic phosphate buffer was added to 5 ml. of Tyrode solution. When bicarbonate buffer was used the ph was varied either by altering the concentration of NaHCO3 at constant C02 concentration, or by altering the concentration of CO2 at constant NaHCOs concentration. Different mixtures of C02 and 02 were provided in cylinders by the British Oxygen Company. The gas was bubbled into the solution, in a beaker fitted with a rubber

2 Ca AND ph IN ANAPHYLAXIS 273 cap, through a hypodermic needle, which was raised above the level of the liquid after addition of the antigen (10-3 egg albumin) to avoid frothing. The histamine content of solutions was measured on the atropinized guinea-pig ileum and is expressed in terms of the content of histamine in the lung particles, which ranged between 7 and 20,Ag of histamine base per gram of tissue. RESULTS The effect of calcium When sensitized guinea-pig lung is placed in Tyrode solution containing antigen, about 20-50% of the tissue histamine is released into the surrounding fluid. When Tyrode solution from which calcium chloride has been omitted is used instead, histamine release with antigen is greatly diminished, and when Tyrode solution is used to which a calcium chelating agent (0.05% versene; ethylenediaminetetraacetate) has been added, histamine release with antigen is reduced to the control level in the absence of antigen (Fig. 1). 30 Tyrode s c 10 Tyrode - Ca Tyrode + Versene Control Fig. 1. Effect of removal of calcium from Tyrode solution on histamine release by antigen from chopped sensitized guinea-pig lung. Release is expressed as a percentage of the tissue histamine. The columns show partial inhibition of histamine release when CaCl2 is omitted from the Tyrode solution and complete inhibition when 0.05% versene is added to the Tyrode solution. In each case the lung was left for a period of 15 min in the modified Tyrode solution followed by a further 15 min period in the presence of antigen. When sensitized lung is placed in saline solution (0-9 % NaCl) containing antigen, very little histamine release occurs, but the anaphylactic mechanism recovers if calcium is added and the degree of recovery depends on the concentration of calcium, as is shown in Fig. 2. Even with 10 mm calcium the

3 274 J. L. MONGAR AND H. 0. SCHILD release is not completely restored to the level in Tyrode solution. This also requires the addition of buffer. Magnesium and potassium ions are, however, not required, as will be shown in the next section. The omission of calcium ions from the solution, without addition of a calcium chelating agent, is usually insufficient to abolish histamine release completely. In four experiments in saline the mean release of histamine with antigen was 8 + 2% of the Tyrode control, whereas in four experiments in saline containing 0.01 % versene, it was only % of the Tyrode control, which is not significantly different from the spontaneous release which occurs in the absence of antigen. o ' 20 E/ I/ 10 Fig. 2. II Calcium concentration (mm) Effect on histamine release by antigen of addition of calcium to 09 % sodium chloride solution. TABLE 1. Effect on anaphylactic histamine release of adding magnesium or potassium ions to sodium chloride solutions buffered to ph by the addition of NaHCO3 01 g/l. Histamine release is expressed as a percentage of the release in Tyrode solution. Each result is the mean of four experiments MgCI2 conen. KCl concn. (g/l.) (g/l.) Histamine release (%) Effect of magnesium and potassium. When magnesium or potassium ions were omitted from Tyrode3 olution, histamine release was in no way affected. In three experiments in which histamine release in Tyrode solution was compared with release in Tyrode solution from which magnesium and potassium ions were omitted, the following results were obtained: histamine release in Tyrode solution, 23, 40 and 47 %; histamine release in Mg- and K-free Tyrode solution, 25, 36 and 46 %. When magnesium or potassium ions were added to

4 Ca AND ph IN ANAPHYLAXIS 275 saline they failed to restore the anaphylactic mechanism. Table 1 shows that the addition of these ions to a buffered solution of sodium chloride, in concentrations equal to or ten times greater than those found in Tyrode solution, does not increase histamine release above that in saline, but if anything depresses it further. Effect of sucrose. When sodium chloride was replaced by sucrose, histamine release was reduced but not abolished provided that calcium was also present. Sensitized lung suspended in buffered isotonic sucrose (with KHCO3, 0 05 g/l.) released no histamine with antigen, but when 1 mm calcium was added to the sucrose solution histamine release by antigen was 32 %, and with 10 mm 42 %, of the Tyrode control (mean of four experiments). Although the anaphylactic mechanism does not function in pure sucrose solution it is not permanently damaged even by prolonged immersion in sucrose. It was found that sensitized lung tissue which was left in sucrose for up to 3 hr reacted normally with antigen when replaced in Tyrode solution. Effect of calcium' lack on histamine release by octylamine and 48/80. Histamine release by organic bases was not affected by lack of calcium. In two experiments in which 0*05 % versene was added to the Tyrode solution, histamine release by 2 x 1o-4 octylamine was 53 and 76 % of tissue content, compared with 62 and 56% in the control. In one experiment with 10-48/80 histamine release was 33 % as against 35 % in the control. Effect of ph on histamine release in anaphylaxis Histamine release in anaphylaxis is greatly influenced by ph, but in investigating this effect it is necessary to avoid extreme changes of ph since these cause a release of histamine by themselves, as is shown in Fig. 3. Within the range of ph from 6 to 8-5, however, 'spontaneous' histamine release is negligible and we have tested the effect of ph on histamine release in anaphylaxis in this range. Fig. 3 also shows the effect of ph on histamine release by antigen from chopped sensitized lung. In this experiment phosphate buffer solutions, where necessary with the addition of HCI or NaOH, were added to Tyrode solution to give ph values ranging from 5-3 to 9 1. In each case the final ph of the solution after incubation with the tissue particles was checked electrometrically. The curve for histamine release by antigen has a maximum at ph 7-8 and a minimum at ph 6*2-6*5. Details of five experiments are given in Table 2. In each the release curve exhibits a minimum in the range ph 6-6-5, and a maximum in the range ph 7-6-7x9. The mean value for the maximum is at 7 75, and for the minimum (in the two best experiments) at 6-3. A change of ph from 6-3 to 7-8 caused a 25-fold increase in histamine release. Above ph 8 the curve shows a distinct downward trend, but this section of the curve could not be explored further in view of the large spontaneous histamine release

5 276 J. L. MONGAR AND H. 0. SCHILD v 4) C '4 I ph Fig. 3. Effect of ph on histamine release in anaphylaxis (phosphate buffer): 0, release from sensitized lung by antigen; 0, release in absence of antigen. TABLE 2. Effect of ph on histamine release by antigen (egg albumin 1 mg/ml.). Each experiment was done on samples of chopped lung from a different guinea-pig Expt. 1 Expt. 2 Expt. 3 Expt. 4 Expt. 5 A - A, " -A N Release Release Release Release Release ph (%) ph (%) ph (%) ph (%) ph (%) * A ph ph Fig. 4. Effect of ph on histamine release in anaphylaxis (bicarbonate buffer). a, changes of ph produced by varying the concentration of bicarbonate; b, proportion ofc02 in 02-CO2 mixture varied.

6 Ca AND ph IN ANAPHYLAXIS 277 occurring above ph 8-5. Nevertheless, there could be no doubt about the significance of the effect; at ph 8*4 histamine release was reduced on the average to 83 % of the maximum. Two further series of experiments, in which bicarbonate instead of phosphate buffer was used, are illustrated in Fig. 4. In one of these the bicarbonate concentration was varied from 0 to 10 g/l. and the buffering produced by aerating the solutions with oxygen containing 5% CO2 (Fig. 4a). In the other series (Fig. 4b) the concentration of bicarbonate was maintained constant at 0-1 g/l. and the ph was varied by bubbling oxygen containing varying amounts of carbon dioxide ( %). The curves obtained with bicarbonate buffer are similar to those with phosphate buffer in having a maximum at ph and a minimum at ph 6-5, but they differ in the lowest ph range. In phosphate buffer, histamine release at ph 5-5 was definitely greater than at ph 6, whereas in bicarbonate buffer the curve shows no upward trend at values below ph 6. The release of histamine in the presence of antigen by phosphate Tyrode solution below ph 6 can be attributed to tissue damage, since a similar release was obtained in the absence of antigen. In Tyrode solution of ph 5f2, produced by bubbling carbon dioxide without bicarbonate, no histamine release occurred either in the presence or in the absence of antigen. Interaction of calcium and ph The effects of calcium and ph on histamine release in anaphylaxis are closely connected. Calcium lack can be compensated for by increasing the ph. Fig. 5 shows the effect of ph in solutions containing saline with and without versene (a and b) and saline with calcium (c). In the complete absence of calcium (0.05% versene) there is no histamine release which could be attributed to antigen, the curve coinciding with the curve of spontaneous release. In calcium-free solutions, but without versene, there is no histamine release at ph 7 but an appreciable release at ph 8. In solutions containing 1 mm calcium there is reduced histamine release at ph 7 but a strong release, comparable to that in Tyrode solution, at ph 7-8. Inhibition in a solution of low ph can be compensated for by increasing the concentration of calcium. Fig. 6 shows the effects of calcium on histamine release by antigen at ph 6 and 8. The calcium concentration in the Tyrode solution was varied from 1 to 40 mm. At ph 8 there was no effect of calcium concentration on histamine release. At ph 6 histamine release was greatly reduced in 1 mm calcium, but in 5-40 mm calcium it was almost equal to the control value at ph 8. Effect of calcium lack and acidity on desensitization It was shown in earlier work that inhibition of histamine release in anaphylaxis may be produced without interference with desensitization. Thus phenol

7 278 J. L. MONGAR AND H. 0. SCHILD 4, 8105 I. co I 'I ph Fig. 5. Curves a, b and c represent histamine release by antigen from sensitized lung: a, saline with 0-05% versene; b, saline alone; c, saline with 1 mm Ca. Curve d, histamine release without antigen. 20._ ph 8 0 ph 6 U I Ca (mm) Fig. 6. Effect of calcium concentration with change in ph. Each point represents the mean of two experiments. In the experiments at ph 6 the bicarbonate of the Tyrode solution was omitted and CO2 5 % % was bubbled through the solution. The tonicity of the solutions with high calcium was adjusted by reducing the NaCl correspondingly. I 40

8 Ca AND ph IN ANAPHYLAXIS 279 blocks histamine release by antigen from sensitized tissue (Mongar & Schild, 1957a), but after removal of the phenol the tissue is completely desensitized. On the other hand, reduction of temperature also blocks histamine release by antigen, but when the tissue is warmed up again it is found to be incompletely desensitized as is shown by the fact that it now releases histamine (Mongar & Schild, 1957b). The following experiments show the effect of calcium lack on desensitization. 50 d e40 e b a b 10 C Versene. Cold Phenol Tyrode Antigen Fig. 7. Histamine release after removal of the inhibitor. In Expts. a, b and c the antigen was present throughout but the inhibitor was removed half way through the experiment. Expt. d was a control showing, first, spontaneous release and then histamine release by antigen in the absence of inhibitor. Antigen was applied to sensitized lung suspended in calcium-free Tyrode solution containing 0-05 % versene and, as expected, it failed to produce a release of histamine. After 15 min the lung was placed in ordinary Tyrode solution containing antigen and it then released a substantial quantity of histamine (30% of control). This experiment is illustrated in Fig. 7 a, along with corresponding experiments with low temperature (7 b) and phenol (7 c). After removal of the block by low temperature a release of histamine occurs of the same magnitude as after removal ofthe block by versene, whereas after removal of the phenol block no residual histamine release occurs. It is thus clear that calcium lack, like low temperature but unlike phenol, causes a partial inhibition of desensitization which results in a release of histamine when the calcium is replaced. Table 3 shows the effects of phenol, versene and acid ph, and their subsequent replacement by normal Tyrode solution, on histamine release by antigen. Lack of calcium combined with 0 05 % versene produced complete inhibition, and 10 mm phenol and a ph of 6-2 caused substantial reduction of

9 280 J. L. MONGAR AND H. 0. SCHILD histamine release. Removal of block by versene was always followed by histamine release; removal of block by acid ph was also followed by histamine release, though not as consistently as after versene; but removal of block by phenol was not followed by histamine release. TABLE 3. Histamine release by antigen in the presence and after removal of various inhibitors. The two columns represent releases in successive 15 min periods, expressed as percentage of control and corrected for spontaneous release Inhibitor Inhibitor present removed Phenol 10 mm i Versene 0.05% Acid ph Phosphate buffer Bicarbonate buffer The release of histamine which occurs when an inhibiting agent is removed is always considerably less than that which occurs when the antigen is added without inhibitor. This difference can be interpreted in two ways: it might indicate a partial desensitization or it might be due to persistent damage by the inhibitor. The following experiments show, however, that none of the inhibitors used produced a persistent damage. After 15 min exposure of sensitized lung to a solution containing 0 05 % versene (but no antigen) a subsequent anaphylactic reaction in Tyrode gave a release of histamine which was 120% of control; similarly, after exposure to a solution of ph 6 (without antigen) the subsequent histamine release was 110 % of control and after exposure to a solution containing 10 mm phenol it was 90 % of control. It thus seems that neither calcium-lack nor a reduction of ph to 6-2 damages the tissue, and that both induce a partial desensitization. Effect of Ca lack and acid ph on oxygen consumption All inhibitors of anaphylaxis previously studied inhibited oxygen consumption as well as histamine release. Calcium lack differs from these in producing a complete inhibition of histamine release in anaphylaxis without inhibiting oxygen consumption. Oxygen uptake was measured by the manometric method. Chopped lung was suspended in calcium-free Tyrode solution in which the bicarbonate was replaced by phosphate buffer at ph 7-6 with and without 0 05 % versene.

10 Ca AND ph IN ANAPHYLAXIS 281 Similar experiments were also carried out in Tyrode containing phosphate buffer at ph 6-2. The results of these experiments are shown in Table 4. Neither calcium lack nor a ph of 6*2 produced an appreciable diminution of the oxygen consumption of chopped guinea-pig lung. TABLE 4. Effect of calcium lack and- acid ph on oxygen uptake of chopped lung tissue Versene Control Ca-free (5 x 10-4) Acid ph Oxygen uptake (Iul./hr/0.2 ml. tissue) DISCUSSION These experiments show that calcium is required for the anaphylactic reaction and that other cations are probably not essential. The release of histamine in the anaphylactic reaction can be completely inhibited by calcium lack. When calcium was omitted from the Tyrode solution histamine release was greatly reduced and when the chelating versene agent was added it was completely abolished. Potassium produced no effect either when omitted from Tyrode solution or when added to saline solution; it thus does not seem to be required in anaphylaxis. Magnesium also produced no effect when it was omitted from Tyrode solution or added to saline. But our experiments do not exclude the possibility that a trace of this ion may nevertheless be required, since complete abolition of histamine release was only obtained by the addition of versene which binds both calcium and magnesium. On the other hand, the negative results obtained by adding magnesium to saline show that magnesium cannot replace calcium. There was some evidence that magnesium, rather than augmenting the effects of calcium, slightly antagonized these, but this antagonism did not increase with the concentration of magnesium and there was no suggestion of a competitive antagonism between the two divalent cations such as was found by Jenkinson (1957) at the neuromuscular junction. Even the sodium ion is not an essential requirement of the anaphylactic reaction since a substantial histamine release was obtainable in sucrose containing only calcium chloride and a trace of potassium bicarbonate. Effect of ph. The effects of change in ph throw further light on the role of calcium in anaphylaxis. Histamine release in anaphylaxis is maximal at ph 7X8 and with physiological calcium concentration is almost completely inhibited at ph 6X2. It is possible, however, to obtain a substantial histamine release at ph 6 by increasing the concentration of calcium to several times the physiological amount. Conversely, by increasing the ph histamine release can 18 PHYSIO. CXL

11 282 J. L. MONGAR AND H. 0. SCHILD be obtained in a solution containing no free calcium ions. The effects of calcium and ph are thus interdependent; in order to preserve the anaphylactic reaction at reduced ph the concentration of ionized calcium must be increased. A possible interpretation of these findings is that the anaphylactic reaction requires bound calcium and that the binding decreases with ph. It is unlikely that calcium is bound to the carboxyl groups of protein, as it is in the Ca-casein complex, for this binding is unaffected by ph in the range 6f3--8&5 (Chanutin, Ludewig & Masket, 1942). It is possible, however, that Ca combines with the imidazole groups of a protein, since the nitrogen atom of the imidazole ring can form complexes with divalent cations (Gurd & Wilcox, 1956) which dissociate at about ph 6 (Tanford, 1952). Mode of action of calcium. Low calcium is known to increase the stability of mitochondria, and the inhibition of histamine release in anaphylaxis might conceivably be due to increase of stability of the mitochondrial particles which contain histamine. This seems unlikely, however, since the action of other histamine releasers such as octylamine and compound 48/80 is unaffected by calcium lack. Another possibility to be considered is that calcium may be required for the union of antigen with antibody. There is, however, no evidence for such an action of calcium from the in vitro studies of Levine et al. (1953a), who showed that the antigen-antibody reaction takes place equally in the presence and in the absence of calcium. A more probable mode of action of calcium is based on an analogy with complement fixation which, as shown by Levine et al. (1953b), requires the presence of calcium. In a previous communication (Mongar & Schild, 1957 b), we have suggested a scheme of the anaphylactic reaction which involves a cellular mechanism which at one stage resembles the complement-fixation reaction. It was suggested that the antigen-antibody complex combines with a heat-labile protein and that this leads to the activation of an unstable enzyme system which is concerned in the process of histamine release. Calcium may be required in this activation in much the same way as it is for the activation of the first component of complement (Levine & Mayer, 1954). This possible role of calcium is indicated below: Antigen-antibody r- -Fd Enzymeprecursor Ca2+ Inactive enzyme Active enzyme Bound histamine Free histamine

12 Ca AND ph IN ANAPHYLAXIS 283 It is supported by the finding that when antigen is added to sensitized lung in the absence of calcium, and calcium is added afterwards, histamine release only occurs when the calcium is added. Nevertheless, the histamine release which occurs under these conditions is not as great as the normal and some explanation is required for an apparent partial desensitization in the absence of calcium. It may, perhaps, be postulated that in the absence of calcium the enzyme precursor is partly converted to an inactive product (indicated by the broken line). An analogy to this is provided by the transformation of trypsinogen, which in the presence of calcium is converted (by the autocatalytic effect of a trace of trypsin) into trypsin but in the absence of calcium is mainly converted into inactive products (McDonald & Kunitz, 1941). Oxygen consumption. In previous work on inhibition of anaphylaxis (Mongar & Schild, 1957a), it was found that all substances which inhibit histamine release in anaphylaxis also inhibited oxygen consumption. Some of the substances tested such as dinitrophenol inhibited oxygen consumption much more than histamine release, others such as antipyretics inhibited both processes in about the same concentrations, but none inhibited anaphylaxis without depressing oxygen consumption. These findings, coupled with the fact that anoxia also inhibits histamine release in anaphylaxis, seemed to throw doubt on the possibility of discovering a selective inhibitor of anaphylaxis, but the fact that calcium lack and acid ph inhibit anaphylaxis but not oxygen consumption shows that it is possible to block the anaphylactic reaction without affecting the cell as a whole. SUMMARY 1. Histamine release in anaphylaxis from chopped, actively sensitized guinea-pig lung is greatly reduced if calcium ions are omitted from the Tyrode solution in which the lung particles are suspended, and is abolished if 0.01 % versene (ethylenediaminetetraacetate) is added. 2. Histamine release by compound 48/80 and octylamine is not reduced by calcium lack. 3. Omission of potassium and magnesium ions does not affect the anaphylactic histamine release and replacement of sodium ions by sucrose reduces but does not abolish it. Addition of calcium ions to a buffered sodium chloride solution restores the anaphylactic histamine-releasing mechanism whereas the addition of magnesium or potassium ions is ineffective. It thus seems that the anaphylactic reaction requires the presence of calcium ions but not magnesium, potassium or even sodium ions. 4. Histamine release in anaphylaxis is ph-dependent. It has an optimum at about 7-8 and falls off at higher and lower values. At ph 6-2 it is almost abolished. 18-2

13 284 J. L. MONGAR AND H. 0. SCHILD 5. The effects of calcium and ph are interdependent. Inhibition by low ph can be counteracted by adding calcium to the solution and inhibition by low calcium can be counteracted by making the solution more alkaline. These findings suggest the formation of a calcium-protein complex which dissociates at an acid ph. 6. Calcium lack not only inhibits histamine release in anaphylaxis but also interferes with desensitization. It is concluded that calcium is probably needed in the activation of an enzyme system required in the anaphylactic reaction. REFERENCES CHANuTiN, A., LUDEWIG, S. & MASKET, A. V. (1942). Studies on the calcium-protein relationship with the aid of the ultracentrifuge. J. biol. Chem. 143, GURD, F. R. N. & Wacox, P. E. (1956). Complex formation between metallic cations and proteins, peptides and amino acids. Advanc. Protein Chem. 11, 34. HUMPHBEY, J. H. & JAQUES, R. (1955). The release of histamine and 5-hydroxytryptamine (serotonin) from platelets by antigen-antibody reactions (in vitro). J. Physiol. 128, JENKINSON, D. H. (1957). The nature of the antagonism between calcium and magnesium ions at the neuromuscular junction. J. Physiol. 138, LEviNE, L., COWAN, K. M., OSLER, A. G. & MAYER, M. M. (1953a). Uptake of complement nitrogen by specific precipitates and its inhibition by ethylene diamine tetra acetate. J. Immunol. 71, LEVINE, L., COWAN, K. M., OSLER, A. G. & MAYER, M. (1953b). The essential role of calcium in complement fixation. J. Immunol. 71, LEVINE, L. & MAYER, M. M. (1954). Kinetic studies in immune haemolysis. V. Formation of the complex EAC'Z and its reaction with C'v,. J. Immunol. 73, McDONALD, M. R. & KuNITZ, M. (1941). The effect of calcium and other ions on the autocatalytic formation of trypsin from trypsinogen. J. gen. Physiol. 25, McINTIRE, F. C., ROTH, L. W. & RICHARDS, R. K. (1949). The in vitro release of histamine from the blood cells of sensitized rabbits: relationship to blood coagulation mechanisms. Amer. J. Phy8iol. 159, MONGAL, J. L. & SoHLD, H. 0. (1956). Effect of antigen and organic bases on intracellular histamine in guinea-pig lung. J. Physiol. 131, MONGAR, J. L. & SCHLD, H. 0. (1957a). Inhibition of the anaphylactic reaction. J. Physiol. 135, MONGAR, J. L. & SCHMLD, H. 0. (1957b). Effect of temperature on the anaphylactic reaction. J. Phy8iol. 135, MONGAR, J. L. & SCHILD, H. 0. (1957c). The need for calcium in the anaphylactic reaction. J. Physiol. 136, 31-32P. RocHA E SILVA, M. (1952). Concerning the mechanism of anaphylaxis and allergy. Brit. med. J. i, TANFORD, C. (1952). The effect of ph on the combination of serum albumin with metals. J. Amer. chem. Soc. 74,