M Rocha e Silva

CHEMICAL MEDIATORS OF THE ACUTE INFLAMMATORY REACTION

We entered the field of mediators of the acute inflammatory reaction in 193841 (Bier & Rocha e Silva, 1938 & 1939; Rocha e Silva & Dragstedt, 1941) immediately after Menkin published his results on the characterization of a new principle to which he gave the name of leukotuxine (Menkin, 1936, 1937 & 1938). Our criticism of the way leukotaxine was introduced in the literature, and especially of the nonreliability of the Trypan blue test, utilized by Menkin to characterize a new substance, was presented recently in a symposium which will be published soon (Rocha e Silva, 1963). According to Menkin, leukotaxine could be sharply distinguished from histamine by three main arguments: (1 1 Leukotaxine was entirely inactive upon the isolated guinea pig ileum. (2) Histamine was unable to attract leukocytes as could leukotaxine by intravenous injection, or when applied to the peritoneal cavity of rabbits in glass tubes with one of the extremities sealed, according to a technique described by Massart and Bordet (1891). (3) When histamine or Ieukotaxine was applied to the rabbit’s skin, and Trypan blue injected intravenously, histamine would produce a different reaction from that of leukotaxine: a blue halo surrounding a whitish central area, though leukotaxine would produce a deeply stained homogenous spot similar to that produced by inflammatory exudates or noxious agents when applied to the skin. In 1939, at the Third International Microbiological Congress, in New York, Menkin presented pictures of the “crystals” of leukotaxine and asked in a rather dramatic way if Bier and Rocha e Silva would think that such crystals were of histamine. This was strongly denied, and it was pointed out that histamine was not in these crystals but presumably in the rabbit’s skin! Though 25 years have passed through the grinding mill of investigations in this field, we still can say emphatically that a positive Trypan blue test should be interpreted with the utmost caution, since the skin of most species utilized for such tests contains and may release histamine. Furthermore, we know that enzymes which release bradykinin and related polypeptides, the kininogenins, also produce a strong blue color under these circumstances. Therefore, the argument conveyed by the absence of inhibitory effects of antihistaminics might not be as strong nowadays as it was a decade ago. Nevertheless, when antihistaminics are effective to totally or partially block the blue test, a histamine mediation can be assumed. To be fair with Menkin, we have to include in the same criticism other factors which have been postulated on the basis of the blue test, and in that line we should mention as one of the most important, the globulin Dilution Permeability Factor (DFP), introduced by Miles and Wilhelm (1955, 1958 & 1960a). This was obtained by diluting with saline the serum of the guinea pig, man, and of other species. This factor might act indirectly by releasing polypeptides of the bradykinin type, since SBTI and DFP are able to block its permeability-increasing effect. This factor can safely be included among the kininogenins which are enzymes that act by releasing kinins from their inactive precursors, kininogens, present in plasma.

THE PHYSIOLOGICAL SIGNIFICANCE OF BRADYKININ

Bradykinin can be included in the group of the so-called tissue-hormones, in the sense that there is no specialized gland to secrete it. Being a polypeptide originated from the a*-globulin of plasma, its presence everywhere in the organism makes it a potential factor in any process in which proteases are activated. But this, we know, will not merit the honorific title of a hormone. We have to find a condition in which the release of bradykinin might explain a genuine physiological phenomenon. I give as an example the case of histamine. In spite of half a century of continuous work, we still have doubts about its importance as a factor in normal physiology. With serotonin, which is about of the same age as bradykinin, we have the same anxiety when asked which role i t plays in normal physiology. A few years ago, with the demonstration by Hilton and Lewis (1956-57) that bradykinin is released by an enzyme secreted by the sub-mandibullary salivary glands to produce the hyperemia which accompanies the work of the gland, we had the inner satisfaction that at least bradykinin, among its sisters, histamine and serotonin, would have a definite role to play in normal physiology. Since salivation is quite physiological, and the hyperemia of the gland a necessary consequence of the work of the gland, then bradykinin would at least help one to understand this modest physiological phenomenon. But, alas! a few years later, Schachter (1960) showed that at least in the guinea pig bradykinin might not play such a role, since the saliva of the guinea pig does not release bradykinin when in contact with the guinea pig plasma. Unless we demonstrate that in the guinea pig there is no hyperemia accompanying the work of the gland, Hilton and Lewis’ point of view is weakened, though not entirely disproved, since we can always assume that the physiology of the guinea pig is a little bit different from that of other species. We know a number of such differences and sometimes I wonder whether the aphorism of Cuvier “tot sunt species quot ab initium creavit infinitum ens,” is not still valid nowadays. But we still had the demonstration by Armstrong et al. (1955-57) that bradykinin or a very similar polypeptide might be one of the physiological transmitters of pain, being fairly well identified with the so-called “painproducing substance” (PPS) obtained when plasma is transferred to uncoated glass containers. But, then, alas! again. Somebody came to say that pain is not physiological, since if everything goes fine we should not feel pain. At this point we are very prone to lose temper and ask what is physiological, after all? How far should go the oscillations of the normal physiological parameters to be called pathological? We’ found, for instance, as we are going to discuss later, that bradykinin is released when the skin is warmed

Potentiation of Bradykinin and Eledoisin by BPF (Bradykinin Potentiating Factor) from Bothrops jararaca Venom.

Nachweis der pharmakologischen Wirkung des Bradykinins am isolierten Meerschweinchenileum und am deutlichen Blutdruckanstieg bei Hund und Katze nach Vorbehandlung mit BPF (Bradykinin-potenzierender Faktor aus Jararcagift). Unter diesen Bedingungen werden mit Bradykinin gleich intensive Effekte wie mit Eldoisin erhalten.

Potentiation of bradykinin by dimercaptopropanol (BAL) and other inhibtors of its destroying enzyme in plasma

Abstract Metal-binding agents such as BAL, versene, thioglycolic acid, cysteine and 8-hydroxyquinoline were found to inhibit the bradykinolytic activity of rat plasma and with the exception of 8-hydroxyquinoline, to potentiate the effect of synthetic bradykinin upon the guinea pig ileum. Our results agree with the suggestion that carboxy-peptidase-B might be the enzyme responsible for the inactivation of bradykinin in plasma. A general similarity seems to exist between inhibition of the bradykininolytic activity and potentiation of the action of bradykinin upon isolated preparations except in the case of 8-hydroxyquinoline. This suggests that the potentiation of bradykinin may be due to inhibition of the enzyme which inactivates it, presumably present in the biological preparations.

Behavioural and somatic effects of bradykinin injected into the cerebral ventricles of unanaesthetized rabbits

1 . The effects of bradykinin (1–5 μg) injected into the cannulated lateral cerebral ventricles were studied in unanaesthetized rabbits before and after intravenous atropine, diphemanil and morphine. 2 . The intraventricular injections of bradykinin produced a short‐lasting phase of behavioural excitation with vocalization followed by sedation. The behavioural excitation was associated with desynchronization in the electrocorticogram (e.co.g.), bradycardia and hypotension followed by tachycardia and hypertension. Tachypnoea was also observed. The subsequent phase of sedation was more prolonged and associated with synchronization of the e.co.g. and signs of catalepsy. Intense miosis was present during both phases. 3 . With repeated intraventricular injections of bradykinin, excitation, miosis, cardiovascular responses and tachypnoea diminished and eventually disappeared but the sedation did not exhibit tachyphylaxis. 4 . Atropine abolished the e.co.g. desynchronization, vocalization and bradycardia, reduced the duration of the excitatory and sedatory phase, diminished the tachycardia and hypotension, enhanced the hypertension, but did not affect the miosis and tachypnoea. 5 . Diphemanil affected only the cardiovascular effects produced by intraventricular bradykinin. They were affected in the same way as by atropine. 6 . Morphine did not affect the excitatory phase, but enhanced the cardiovascular effects produced by intraventricular bradykinin. 7 . The intraventricular injection of bradykinin (50 μg) caused a reduction in the amount of noradrenaline but not of 5‐hydroxytryptamine (5‐HT) in the brain stem; the amount of dopamine in the caudate nuclei was not affected. 8 . It is suggested that central cholinergic and adrenergic systems are activated by intraventricular bradykinin.

Metabolic studies on the release of histamine by compound 4880 in the rat diaphragm

Abstract The inhibitory action of uncoupling agents like 2:4-dinitrophenol, salicylate, thiopental and sodium oleate on the release of histamine from isolated rat diaphragm by compound 48 80 , is markedly decreased by glucose. Inhibitions due to sodium cyanide or anoxia are similarly affected. Rat mesentery mast cell damage by 48/80 in vitro, which is suppressed by dinitrophenol or sodium cyanide, will occur if the action of these inhibitors is tested in the presence of glucose. Sodium succinate cannot prevent the inhibition of histamine release by dinitrophenol, although oxygen consumption studies reveal that it is aerobically metabolized by dinitrophenol-treated tissue. The results obtained have been interpreted as an indication that metabolic intermediates necessary for the release of histamine from rat diaphragm can be generated by a mechanism, possibly glycolytic, functioning independently of the Krebs cycle.

A brief survey of the history of inflammation

It is a heavy task to trace the history of any branch of science, and certainly the most difficult when we try to describe the last segment, that is, the present phase of that history, when the protagonists of the history are still alive; and even worse if they are assembled in the same room, as are many of them present here in this inaugural session of a Symposium on Inflammation. We could not assemble all of them, because of the extension of the subject, the distance to be travelled, from Europe, Canada, Australia, Japan, and the USA, and worst of all, because of the terrible rate of exchange of our cruzeiro toward the dollar. But, none the less, we have the pleasure and honor to be host to distinguished representatives of the basic science in one of the most basic subjects in medicine, namely the Mediators of Inflammatory Reactions. But to trace the history of what is going on in any chapter of experimental medicine is a most difficult task indeed. It is easy to say that Celsius in the ist century A.D. was the first one to describe the main four signs of inflammation: rubor (redness), tumor (swelling), with calor (heat) and dolor (pain), which is well represented in the ex-libris of the Inflammation Club, and I present here the popular picture, usually attributed to Willoughby (Fig. 1). Through his popular drawing we can see that even today what we are trying to prove scientifically was already contained in the profound intuition of a Roman doctor 2000 years ago.

THE ROLE PLAYED BY LEUCOCYTES AND PLATELETS IN ANAPHYLACTIC AND PEPTONE SHOCK

If one goes back to the earlier theories of anaphylaxis, the number of separate points of view, or schools, defending entirely independent ideas on the mechanism of this important phenomenon becomes quite impressive. We might distinguish among the different conceptions-the immunological, the hematological, and the physiological or pharmacological-according to the main principles involved and the personal interests and scientific outlook of their proponents. Today, it is rather trivial to say that the liberation of histamine plays a major role in the production of the symptoms and that other metabolites, such as adenosine, acetylcholine, a “slowly reacting substance” (S.R.S.), etc., might also contribute to initiate or aggravate shock. There is also no question that heparin, as such, is released from the liver and that i t could explain incoagulability of the blood, and that the opposite effect (Le . , the decrease in clotting time as it has been observed in anaphylactic and peptone shock during the so-called negative phase) might depend upon activation or release of thromboplastic agents, among which one might include activation of a proteolytic enzyme of the trypsin type. It is obvious, however, that this description does not include many other phenomena that occur in anaphylactic and peptone shock with at least an equal degree of frequency. I pass over the immunological point of view, since there is a general agreement that the whole anaphylactic reaction takes place as a consequence of a combination of the antigen with the antibody, this being a sort of trigger mechanism for the whole physiological process to develop. The fact that similar phenomena and symptoms can be obtained by injecting peptone led to the conclusion that the symptoms following combination of the antigen with the antibody are common to other forms of aggression to the cell and by no means represent the direct, toxic effect of the so-called antigen-antibody complex. Therefore, reconciliation of the physiological or pharmacological point of view with the immunological one is much easier than might appear by reading papers from either side. First of all, I wish to mention the old French conception of the “colloidoclastic shock,” which implies a disturbance of the colloidal equilibrium of the blood, with consequent fall in leucocytes and platelets, as the main reason for the symptoms observed during anaphylactic and peptone shock. I never understood what point was involved in the idea of the disturbance of the colloidal equilibrium of the blood. There is no question, however, that something remained from this old conception. This refers to the fall in leucocytes and platelets as a characteristic feature of anaphylactic and peptone shock. More detailed studies have subsequently shown that a fall in platelets is one of the most constant indications of a state of sensitization when the antigen i s injected, a t least in certain species of animals. In 1924, Webb’

Comparative pharmacological actions of bradykinin and related kinins of larger molecular weights

Abstract Three polypeptides with the terminal sequence of bradykinin: Gly-Arg-Met-Lys-Bk (GAML-Bk), Met-Lys-Bk, Lys-Bk (kallidin) and bradykinin itself were assayed for biological activity on: guinea pig ileum, rat uterus, rat duodenum, arterial blood pressure of the rat by venous and arterial routes, and vascular permeability (blue test). The pharmacological actions were qualitatively similar but differed quantitatively according to the molecular weights of the peptides tested. Those with the highest molecular weights were much less active upon the different smooth muscle preparations, while 10–12 times more potent in the vascular permeability test. The effects on the rat duodenum and rat uterus ran parallel, but again were discrepant when compared with the guinea pig ileum. The nature of the receptors for bradykinin is discussed.

Anti-inflammatory action of sulfated polysaccharides.

Abstract A number of sulfated polymers: pentosan polysulfate, polyvinyl sulfate, polyethylene sulfonate, cellulose sulfate, dextran sulfate, sulfate amylopectin, in the same way as carragenin, degraded carragenin and dextran, produce edema in the rats paw by local injection, to release kinins from fresh guinea pig and rats plasma (in vitro) and to reduce the bradykininogen content of rats blood when injected i.v. When given repeatedly by the i.p. route, cellulose sulfate and sulfated amylopectin reduced or abolished the edema produced in the rats paw by a subsequent local injection of the same or other edematogenous polymers. This cross desensitization or anti-inflammatory action of the sulfated polymers was also observed if the animals received locally a fraction of the venom of Agk. piscivorus, which was found to split the ester bond of BAEE and release kinin when incubated with fresh plasma of different species. The possible significance of these findings in order to derive a new class of anti-inflammatory agents is discussed.