Pulmonary intravascular sequestration of activated neutrophils: failure to induce light-microscopic evidence of lung injury in rabbits.

Rabbits were injected intravenously with glycogen-elicited allogenic peritoneal polymorphonuclear neutrophil leukocytes (PMNs) for the study of the light-microscopic effects of acute and chronic sequestration of PMNs in the pulmonary vascular bed. Infusion of 51Cr-labeled PMNs demonstrated that approximately half of the cells were sequestered in the lung, with no difference observed between PMNs incubated with 10% normal rabbit serum and PMNs incubated with 10% zymosan-activated serum (ZAS) prior to infusion. Quantitative histologic studies demonstrated that the number of ZAS-activated PMNs present in the alveolar walls at 4 hours rapidly declined over the ensuing 20 hours and was back to buffer control values by 48 hours. No PMNs, red cells, or signs of edema were visible in the alveolar spaces. In rabbits injected chronically (twice weekly for 8 weeks) with 2 x 10(8) PMNs (ZAS-stimulated and unstimulated), no qualitative or quantitative (mean linear intercept) evidence for damage to alveolar walls was observed. These studies indicate that acute and chronic pulmonary sequestration of PMNs activated in vitro, infused in the absence of activated serum products, does not cause light-microscopic evidence of lung injury.

Extra- and intracellular Ca2+ requirements for lysosomal enzyme secretion in human neutrophils.

Ca2+-EGTA combinations were utilized in Hank's buffers to fix extracellular free Ca2+ concentrations [Ca2+f] for the study of human neutrophil lysosomal enzyme secretion. Ca2+-dependent neutrophil secretion initiated by formyl-methionyl-leucyl-phenylalanine (FMLP) and C5a required small amounts of [Ca2+f] (1-3 x 10(-6) M) while that caused by ionophore A23187 required 10(-5) M or greater [Ca2+f]. The inhibition of FMLP- and C5a-induced lysosomal enzyme secretion by the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) was additive to lowering extracellular [Ca2+f] from 10(-4) M to 10(-6) M or blocking plasma membrane Ca2+ flux with verapamil. These results suggest that extracellular and intracellular Ca2+ flux may be coupled in the initiation of neutrophil secretion caused by FMLP and C5a.

Thromboxane B2 and prostaglandin E2 in the rat kidney with unilateral ureteral obstruction.

The production of prostaglandin E2- (PGE2) like and thromboxane A2-(TXA2) like substances is increased after release of unilateral ureteral obstruction (UUO) for 3 days in the isolated perfused rabbit kidney. It has been postulated that this increase in TXA2 biosynthesis might contribute to the development of vasoconstriction in the obstructed kidney. In the present studies, the production of TXA2 and PGE2 in the kidney was further investigated in rats after UUO for 2-18 h. Radioimmunoassay was used to determine thromboxane B2 (TXB2), a chemically stable metabolite of TXA2, and PGE2 production during the incubation of renal slices in vitro. Unlike previous studies, an increase in TXB2 and PGE2 production was demonstrable in the obstructed kidney even in the absence of pharmacological stimulation by bradykinin or angiotensin II. The effect of UUO on prostaglandin production differed in the different anatomical parts of the kidney. In the papilla, production of both TXB2 and PGE2 was increased in the obstructed kidney. In the cortex, however, UUO had a stimulatory effect only on TXB2 production but not on PGE2 production. The increase in TXB2 and PGE2 production was demonstrable as early a 2 h (tested) after ureteral obstruction. Prolongation of ureteral obstruction for 18 h diminished the stimulatory effect of UUO on PGE2 production but not on TXB2 production.

Requirements for different Ca2+ pools in the activation of rabbit platelets. I. release reaction and protein phosphorylation.

We examined the role of Ca2+, both extracellular and intracellular in origin, in the release reaction and protein phosphorylation in rabbit platelets stimulated with platelet activating factor (acetylglyceryl ether phosphorylcholine), thrombin, or ionophore A23187. In the presence of extracellular Ca2+, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca2+ transport, blocked platelet activating factor-initiated serotonin release at a half-maximal inhibitor concentration of 40 microM, compared to 350 microM for thrombin-induced release and greater than 500 microM, for A23187-induced release. Platelet activating factor-induced phosphorylation of two platelet proteins of Mr = 41,000 (P7P) and 20,000 (P9P) was inhibited by TMB-8, an effect which was additive to that caused by removing extracellular Ca2+. TMB-8 demonstrated only minor to non-existent inhibitory effect on phosphorylation in thrombin- or A23187-stimulated platelets. In contrast to P9P phosphorylation, phosphorylation of P7P caused by platelet activating factor was more dependent on a TMB-8 sensitive step than on the availability of extracellular Ca2+. Experiments with buffers containing fixed concentrations of free Ca2+ revealed that both processes (release and phosphorylation), when stimulated by platelet activating factor and thrombin, had the same threshold requirement (1-3 microM) for extracellular free Ca2+. These studies provide evidence tht stimulation of rabbit platelets by platelet activating factor is more dependent on a TMB-8-sensitive intracellular Ca2+ source than is stimulation caused by thrombin. Furthermore, our data indicate that activation of different intracellular processes involved in platelet secretion (such as P7P and P9P phosphorylation) may require Ca2+ from different pools.

Requirement for different Ca2+ pools in the activation of rabbit platelets II. Phospholipase activity.

The effects of extracellular Ca2+ concentration and the putative antagonist of intracellular Ca2+ movement, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on platelet phospholipase activity and thromboxane B2 synthesis were examined in rabbit platelets stimulated by platelet activating factor, thrombin and ionophore A23187. TMB-8 markedly inhibited the platelets stimulated by platelet activating factor, thrombin and ionophore A23187. TMB-8 markedly inhibited the platelet activating factor-induced decrease in [14C]arachidonate content in platelet phosphatidylcholine and phosphatidylinositol, while showing minimal effects on thrombin-induced phospholipase activation. A23187 stimulation of these processes was inhibited to an intermediate degree by TMB-8. In contrast, extracellular Ca2+ removal inhibited phospholipase activity to a similar degree with all three stimuli. Moreover, the threshold concentration of extracellular Ca2+ for phospholipase activation, as measured by thromboxane B2 synthesis, was similar for platelet activating factor- and thrombin-stimulated platelets. These data provide evidence that, while platelet activating factor and thrombin may, to some extent, have similar requirements for extracellular Ca2+, they utilize a TMB-8 sensitive step to different degrees during activation of platelet phospholipase.

Activation of human neutrophils with 1-O-hexadecyl/octadecyl-2-acetyl-sn-glycerol-3-phosphorylcholine (platelet activating factor).

1-O-Hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), the acetylated alkyl phosphoglyceride known as platelet-activating factor, stimulated human neutrophil (PMN) exocytosis, migration, superoxide production and aggregation over a concentration range of 10(-10) to 10(-5) M. AGEPC-induced PMN exocytosis of azurophilic (myeloperoxidase and beta-glucuronidase) and specific (lactoferrin and lysozyme) lysosomal granules was rapid (T 1/2 = 20 sec), dependent on the presence of cytochalasin B, but was not associated with release of cytoplasmic LDH. As seen with the complement-derived peptide stimulus, C5a, AGEPC-initiated PMN enzyme release was dependent on temperature and cellular glycolysis but not on the presence of extracellular Ca++. When analyzed by gradient analysis, PMN migration caused by AGEPC was primarily chemotactic in nature. An unusual feature for both enzyme secretion and migration was a decrease in response between 10(-6) M and 10(-5) M AGEPC. This decreased responsiveness could be explained by rapid PMN desensitization occurring at high AGEPC concentrations, limiting the overall cellular response. Rapid desensitization for exocytosis was demonstrated in PMN stimulated with AGEPC in the absence of cytochalasin B. When cytochalasin B was added subsequently and PMN challenged with AGEPC or C5a, stimulus-specific desensitization to AGEPC but not C5a-induced lysosomal enzyme release occurred. PMN desensitized to C5a responded normally to a subsequent AGEPC challenge. Stimulation of all the PMN functions examined was markedly attenuated with removal of the 2-acetyl group from AGEPC (lyso GEPC). These results suggest that AGEPC stimulates a wide variety of human PMN responses by a receptor-like mechanism, dependent on the short chain fatty acid ester in the 2-position of the alkyl phosphoglyceride.

Effects of extracellular Ca2+ and the intracellular Ca2+ antagonist 8-(n,n-diethylamino)octyl-3,4,5-trimethoxybenzoate on rabbit platelet conversion of arachidonic acid into thromboxane.

The regulatory role of Ca2+ on the conversion of arachidonic acid (AA) into thromboxane B2 (TXB2) was examined in washed rabbit platelets, whose secretory processes are known to have requirements for extracellular Ca2+. Varying the extracellular free Ca2+ [Caf2+] concentration from less than 10(-8) M to 10(-3) M had no significant effect on the synthesis of immunoreactive TXB2 by rabbit platelets incubated with 1-4 microM AA. On the other hand, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca2+ movement, inhibited AA-stimulated synthesis of TXB2 in a concentration dependent manner--an effect which could be partially overcome by increasing the AA concentration. The TMB-8 inhibition could not be reversed by increasing the [Ca2+f]. Studies examining platelet metabolism of 14C-AA and 14C-prostaglandin H2 demonstrated that TMB-8 inhibited platelet cyclooxygenase, but not thromboxane synthetase. These studies demonstrate the absence of a requirement for [Ca2+f] but suggest the presence of a TMB-8 sensitive intracellular Ca2+ pool in the rabbit platelet synthesis of TXB2 from AA.

Quantitation and stability of the fifth component of complement (C5) in bronchoalveolar lavage fluids obtained from non-human primates.

The functional activity and antigenic expression of the fifth component of complement (C5) in concentrated bronchoalveolar lavage fluids (BALF) and serum samples obtained from 13 nonsmoking and 13 smoking baboons was quantified by specific hemolytic titration and radioimmunoassays. The number of C5H505 units per milligram of albumin in BALF obtained from nonsmoking and smoking baboons indicated a nearly 2-fold increase in C5 hemolytic activity in lavage fluids as compared to matched serum samples. No significant differences (p greater than 0.05) in C5 hemolytic or antigenic (radioimmunoassay) expression were evident between BALF samples obtained from nonsmoking and smoking baboons. C5 stability in BALF was indicated by the highly significant correlation (p less than 0.005) between C5 hemolytic activity and antigenic expression. The absence of C5-cleaving enzymes in BALF was verified by incubation of 125I-C5 with these fluids, followed by SDS5 polyacrylamide slab gel and autoradiographic analyses. These results demonstrated the presence and stability of substantial quantities of functionally active C5 in the secretions of primate lungs, which represents a readily available source of phlogistic fragments for the initiation of pulmonary inflammation.

Thromboxane B2 and prostaglandin E2 in the K+-depleted rat kidney.

There is a considerable amount of interest in prostaglandin E2 (PGE2) metabolism in potassium depletion, but the findings remain inconclusive. Thromboxane A2 (TXA2) is another type of prostaglandin with a vasoconstrictive property and its biosynthesis in the kidney is altered under pathophysiological conditions. We investigated the production of both immunoassayable PGE2 and thromboxane B2 (TXB2), a chemically stable metabolite of TXA2, in the chronically K+-depleted rat kidney. During a 90-min in vitro incubation of papillary slices obtained from K+-depleted rats, TXB2 production was increased, but PGE2 biosynthesis was decreased and PGF2 alpha remained unaltered compared with control rats. In the cortex, TXB2 production was low, but it was greater in K+-depleted rats compared with control rats. Deletion of K+ from the incubation medium had no measurable effect on either TXB2 or PGE2 production in both K+-depleted and control rats. Formation of [14C]TXB2 from [14C]PGH2 by microsomes from renal papilla was greater in K+-depleted rats compared with control rats, suggesting that the increased TXB2 production in the K+-depleted rat kidney is probably due to an activation of TXA2 synthetase.

Effects of C5a on baboon alveolar macrophage migration.

The effect of C5a, a fragment of the fifth complement component, on alveolar macrophage migration was studied using a micropore method. Cells were obtained by broncho-pulmonary lavage, in vivo or in vitro, from control baboons and baboons taught to smoke cigarettes. Macrophage migration directly depended on the C5a concentration cells encountered. The highest C5a concentrations studied enhanced migration more than 20-fold. Cells from smokers exhibited greater migration than those from nonsmokers, and cells recovered in vivo exhibited greater migration than in vitro cells, but these differences were not significant. A gradient analysis suggested that C5a-induced migration was chemotactic, i.e., oriented; C5a enhanced migration more than N-formyl-methionyl-phenylalanine, and C5a without the C-terminal arginine lost over two thirds of its capacity to enhance migration. The observation that C5a induces alveolar macrophage chemotaxis established that alveolar macrophages are similar to other mononuclear phagocytes with respect to C5a-enhanced migratory responses, and demonstrated that complement components may participate in lung inflammatory reactions through effects on cells other than neutrophils.

Activation of rabbit platelet phospholipase and thromboxane synthesis by 1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor).

1-O-Hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), structurally identical with platelet activating factor, is a potent stimulus for rabbit platelet aggregation and serotonin secretion. AGEPC at concentrations between 10(-10) and 10(-8) M induced stimulation of rabbit platelet synthesis of thromboxane B2. The dose vs. response curve for platelet thromboxane B2 synthesis was displaced slightly towards higher stimulus concentrations compared to [3H]serotonin secretion, with half-maximal concentrations of 2.5 . 10(-9) and 8 . 10(-10) M, respectively. Rates of thromboxane B2 synthesis and secretion were similar with a t 1/2 max of 4.0-4.5 s for both processes. AGEPC induced a decrease in platelet [14C]arachidonic acid in both phosphatidylinositol and phosphatidylcholine, although [14C]arachidonic acid turnover in phosphatidylcholine was not observed below 1 . 10(-8) M AGEPC. Concomitantly, this decrease in phospholipid [14C]arachidonic acid was associated with a marked increase of radiolabel in platelet diacylglycerol and phosphatidic acid 15 s after AGEPC addition, suggesting the possibility of a phospholipase C-diacylglycerol lipase mechanism of fatty acid cleavage. As observed previously with secretion and aggregation, removal of the 2-acetyl group from AGEPC abrogated all capacity of this molecule to stimulate platelet phospholipase. This study indicates that AGEPC (or platelet activating factor) activation of rabbit platelet phospholipase occurs in a time-course and concentration range similar to that required for [3H]serotonin secretion.

Leukocytes in chemotactic-fragment-induced lung inflammation. Vascular emigration and alveolar surface migration.

Lung inflammation was induced in rabbits by intratracheal injections of chemotactic fragments obtained from zymosan-activated serum (CF-ZAS), and the route of vascular emigration and alveolar surface interaction of polymorphonuclear leukocytes (PMNs) and monocytes migrating into the lung was characterized by transmission (TEM) and scanning (SEM) electron-microscopic examination. Leukocytes migrated from capillaries and venules into the alveolar wall interstitium by adherence to the vascular endothelium and migration through the endothelial intracellular junction to attain a position between a reapposed endothelial cell junction and the vascular basement membrane. The cells then migrated into the interstitium through a narrow opening in the basement membrane. Leukocyte entrance into the alveolar space from the interstitium appeared to occur through small openings in the epithelial basement membrane at or near the Type I epithelial intercellular junction. Once in the alveolus, PMNs and macrophages demonstrated surface adherence and spreading along with evidence of migration, pseudopod extension, interalveolar pore transit, and retraction fiber formation. This study indicates the leukocyte influx into the alveolus in acute chemotactic-factor-induced inflammation is via a continuum of migrational activity, beginning at the pulmonary capillary endothelial surface and persisting on the alveolar epithelial surface.

Lung inflammation induced by complement-derived chemotactic fragments in the alveolus.

The intratracheal injection into rabbits of low molecular weight C5-derived chemotactic fragments (C5fr), prepared from zymosan-activated serum, induced an acute pulmonary inflammation characterized by intraalveolar accumulation of neutrophils, erythrocytes, and edema fluid. In separate experiments, depletion of circulating pulmonary neutrophils and absorption of C5fr with immobilized antibody to homogenous human C5a prevented the observed inflammatory changes, indicating a requirement for these two elements in initiating this reaction. When examined by transmission and scanning electron microscopy, lungs of C5fr-injected rabbits revealed pulmonary neutrophils, often appearing partially degranulated, in alveolar, pulmonary capillary, and interstitial spaces. Alveolar spaces and, less often, the interstitial compartment contained fibrinoid deposits with leukocytes and erythrocytes enmeshed in the fibrin strands. Injury to the pulmonary vascular endothelium consisted of bleb formation in capillaries and endothelial basement membrane separation with subendothelial accumulation of inflammatory cells in venules. Type I, but not type II, epithelial cell damage included blebbing and epithelial cell basement membrane detachment. Endothelial and epithelial layer damage was always associated with pulmonary neutrophils continguous to the injured structures. These studies indicate the potential for alveolar epithelial and capillary injury in neutrophil-associated pulmonary inflammation resulting from intraalveolar accumulation of chemotactic substances.

The binding of rabbit basophil-derived platelet-activating factor to rabbit platelets.

The platelet binding characteristics of platelet-activating factor (PAF), a basophil-derived lipid that causes aggregation and secretion in rabbit platelets, were studied. In an assay in which binding was quantitated by loss of PAF activity, suspensions of washed rabbit platelets rapidly removed PAF from solution. Rabbit erythrocytes, lymphocytes, and neutrophils also bound PAF, but their binding capacity was less than that of the platelets. PAF binding to rabbit platelets was saturable and dependent on temperature and concentration of PAF and platelets but not on the presence of extracellular Ca2+. Initial rates of PAF binding and platelet secretion were identical, although temperature dependence studies suggested that binding was not the rate-controlling step in PAF-induced platelet secretion. Analysis of binding kinetics suggested that a PAF concentrations saturable for platelet secretion, only some of the available platelet binding sites were occupied.

Interaction of Ca2+ and protein phosphorylation in the rabbit platelet release reaction.

Ca2+ flux and protein phosphorylation have been implicated as playing an important role in the induction of the platelet release reaction. However, the interactions between Ca2+, protein phosphorylation, and the release reaction have been difficult to study because secretion in human platelets is independent of extracellular Ca2+. Thus, we studied rabbit platelets, which, unlike human platelets, require extracellular Ca2+ for serotonin release to occur. Thrombin, basophil platelet-activating factor (PAF), or ionophore A23187 treatment of intact 32PO43--loaded rabbit platelets resulted in a 200-400% increase in phosphorylation of P7P and P9P, respectively. These peptides were similar in all respects to the peptides phosphorylated in thrombin-treated human platelets. When Ca2+ was replaced in the medium by EGTA, (a) thrombin- and PAF-induced rabbit platelet [3H]serotonin release was inhibited by 60-75%, whereas ionophore-induced release was blocked completely; (b) thrombin-, PAF-, or ionophore-induced P9P phosphorylation was inhibited by 60%; and (c) ionophore-induced P7P phosphorylation was decreased by 60%, whereas that caused by thrombin or PAF was decreased by only 20%. At 0.25-0.5 U/ml of thrombin, phosphorylation preceded [3H]serotonin release with the time for half-maximal release being 26.0 +/- 1.3 s SE (n = 3) and the time for half-maximal phosphorylation being 12.3 +/- 1.3 s SE (n = 3) for P7P and 3.7 +/- 0.17 s SE (n = 3) for P9P. P9P phosphorylation was significantly inhibited (P less than 0.015) by removal by Ca2+ from the medium at a time point before any thrombin- or ionophore-induced serotonin release was detectable. Thus, our data suggest that Ca2+ flux precedes the onset of serotonin secretion and that the rabbit platelet is an appropriate model in which to study the effects of Ca2+ on protein phosphorylation during the platelet release reaction.

Complement fragments, alveolar macrophages, and alveolitis.

Mechanisms of neutrophil infiltration into the rabbit alveolus have been investigated. Complement activation in the circulation induced pulmonary vascular margination but not a significant level of alveolar infiltration. Instillation of C5 fragments into the airways, however, attracted neutrophils into the alveolar airspaces. The anaphylatoxin-inactive fragment of C5, C5a des Arg, was found to be much more active in this regard than C5a. Furthermore, these fragments were shown to induce the production of a neutrophil-directed chemoctactic factor from pulmonary macrophages, raising the question of whether the C5a des Arg was acting directly to attract neutrophils or indirectly via the macrophage. To substantiate a possible role for C5 and C5 fragments in alveolitis, active C5 was demonstrated in lavage fluids, and macrophage-derived C5 cleaving enzymes have been described. Finally, a route of neutrophil infiltration via migration through the alveolar capillary wall into the interstitium is proposed, and subsequent penetration of the alveolar epithelium out into the airspace. (Am J Pathol 97:93--110, 1979).

Macrophage-mediated tumor cell killing: lack of dependence on the cyclooxygenase pathway of prostaglandin synthesis.

M phi obtained directly from disaggregated murine Moloney sarcomas produced PGE2 and a hydroxy fatty acid derivative as the major products of arachidonic acid metabolism. M phi-immunoreactive PGE synthetic rates decreased substantially and cytotoxic activity was lost when freshly explanted tumor M phi were held in culture 24 hr. Such cultured M phi remained in a partially activated "primed" state, however, wherein the addition of minute (ng) amounts of bacterial lipopolysaccharide (LPS) returned cytolytic activity and PGE synthesis to original levels. Indomethacin-induced blockade of the M phi cyclooxygenase pathway inhibited PG synthesis by LPS-stimulated, primed M phi without affecting the return of cytolytic activity. We conclude, therefore, that the production of PG had no direct role in the mediation of tumor cell killing by activated M phi isolated from these neoplasms.