Two patterns of early neutrophil accumulation in acute inflammatory lesions

A model of acute inflammation in the skin of sheep was developed using¹¹¹In]oxine-labeled neutrophils to quantify the accumulation of neutrophils in inflammatory lesions. Peak accumulation occurred during the first hour in lesions induced by zymosan-activated sheep plasma (ZAP, 100%) and during the second hour in lesions induced by endotoxin (10^–11 moles) and casein (2 x 10^–8 moles). The accumulation of neutrophils after the final stimulus diminished when lesions were restimulated every 2 h on two or more occasions. Thus desensitization of inflammatory lesions previously observed in rabbit skin is not restricted to that species. The early accumulation of neutrophils in primary and restimulated lesions was measured in lesions from 10 min to 2 h old. In primary lesions, ZAP induced significant neutrophil accumulation by 10 min, whereas 30 to 45 min elapsed before significant neutrophil accumulation occurred in lesions induced with endotoxin and casein. The initial responses in restimulated lesions were not diminished, but there was a failure of neutrophil accumulation to continue for as long as occurred in primary lesions. This finding indicates that regulation of the neutrophil influx into dermal inflammatory lesions may occur at a point after occupancy of the putative tissue receptors in the pathway which leads to accumulation of neutrophils at the injection site. The results indicate that there are at least two patterns of neutrophil accumulation in primary inflammatory lesions. The delayed pattern of neutrophil accumulation induced by casein and endotoxin resembles the enhanced binding of neutrophils to stimulated endothelial cultures in vitro, whereas the basis for the immediate response induced by ZAP remains conjectural. It is proposed that the primary action of chemotactic agents may be to provoke changes in the adhesive properties of endothelium and resident connective tissue cells thus permitting the migration of neutrophils into inflammatory lesions.     

Increased expression of CR3 (C3bi receptor) on neutrophils in human inflammatory skin reactions

To help determine whether the neutrophils (PMN) found in skin inflammatory reactions are activated, we have compared the expression of the C3bi receptor (CR3) on such cells with that on autologous blood PMN in 10 pollen-sensitive subjects. Using skin chambers overlying denuded blister bases we collected PMN at 2 or 4 hr at sites of challenge with pollen antigen or buffer solution. These cells and PMN in autologous blood were incubated with monoclonal anti-CR3 antibody and the expression of CR3 was measured by indirect fluorescence and flow cytometry. Significantly more PMN were found at antigen than at buffer sites at 2 hr (7.02±0.45 × 10⁵ vs 0.71±0.25×10⁵) and at 4 hr (2.2±0.57×10⁶ vs 5.47×10⁵). The mean CR3 expressions on PMN at antigen and buffer sites were similar (117±7.4 vs 118±9.0); both were significantly greater than on blood PMN (17.6±1.5;P<0.005). PMN from both sites could be stimulated furtherin vitro with formyl-methionyl-leucyl-phenylalanine (FMLP) to express more CR3 to a level even greater than in FMLP-stimulated blood PMN (155±11 and 157±12, respectively, vs 108±7 in blood PMN). The incubation of blood PMN with the noncellular component of the chamber fluid led to a moderate (28–100%) increase in CR3 expression, but far less than the CR3 expression on the chamber fluid PMN themselves. Since surface CR3 is thought to be an activation marker important in PMN adhesion, these findings may be important in understanding the emigration of PMN in skin inflammatory reactions.     

The effect of vasodilator prostaglandins on polymorphonuclear leukocyte infiltration and vascular injury.

Some severe acute inflammatory reactions are characterized by polymorphonuclear leukocyte (PMN) infiltration as well as vascular and tissue damage with hemorrhage. Two types of mediators that may be involved in such reactions are chemotactic factors and prostaglandins. The chemotactic factors can induce PMN infiltration, while some types of prostaglandins cause vasodilatation. We reported previously that injection of soluble, nonphagocytosable chemotactic stimuli, zymosan-activated plasma (ZAP), or C5a des Arg into rabbit skin induced PMN-dependent hemorrhage. Here we investigated whether prostaglandins may modulate the rate of PMN infiltration, measured with 51Cr-labeled leukocytes and the degree of hemorrhage, measured with 59Fe-labeled red cells. Prostaglandin (PG) E1 (0.5 microgram) or E2 (1 microgram) increased ZAP-induced PMN accumulation by 81% and hemorrhage by 400%. A similar potentiation by PGE2 was observed when submaximal concentrations of ZAP were injected. Prostaglandin F2 alpha had no such effect. These results indicate that the degree of PMN infiltration of the tissues may be one factor determining the severity of vascular damage. Furthermore, vasodilatory prostaglandins, generated during neutrophilic inflammatory reactions, may enhance chemotactic-factor-mediated PMN infiltration and increase the extent of vascular injury.     

The inflammatory reaction in healing wounds: the role of polymorphonuclear leucocytes

The inflammatory process in granulation tissue in full-thickness skin wounds was studied and the role of polymorphonuclear leucocytes (PMNLs) in this process evaluated in an experimental model in the rat. The number of PMNLs in the wound, assessed by determination of the PMNL-specific enzyme myeloperoxidase (MPO) activity in wound exudate, increased from 0.45 U/ml on day 1 after wounding to 0.8 U/ml on day 2, and then remained constant throughout the five days of observation. The concentration of prostaglandin E2 (PGE2) in wound exudate increased progressively from 70 ng/ml on day 1 to 290 ng/ml on day 5. The lack of correlation between these two variables indicated that PMNLs were not the major source of PGE2. Blood flow and albumin extravasation in the granulation tissue were measured, and the relation between these two variables and PMNL accumulation was studied. Rats were rendered neutropenic with an antineutrophil serum, resulting in an 83% decrease in circulating PMNLs and a 61% decrease in granulation tissue MPO activity on day 5, as compared with rats treated with normal rabbit serum. These reductions did not, however, affect either blood flow or albumin extravasation, and no correlation was observed; but when inter-individual variations in the absolute levels of the variables measured were eliminated by calculating in each rat a left-to-right wound ratio, PMNL accumulation correlated well to both blood flow (R = 0.81) and albumin extravasation (R = 0.65). It is suggested that blood flow and albumin extravasation in the granulation tissue are influenced by local PMNL accumulation and, further, that the inflammatory response varies considerably between one animal and another.     

Azathioprine reduces extravasation and neutrophil trafficking in immune complex-mediated inflammation in the rat colon

Azathioprine (AZ) has been used in the treatment of refractory inflammatory bowel disease. The mechanism by which AZ decrease colonic inflammation is not known. It is alluded that AZ may be effective in the maintenance of remission. We examined whether AZ in non-immunosuppressive doses reduces extravasation and neutrophil trafficking in a rat model of colonic inflammation. Rats were treated with I.P. injection of AZ (1 mg/kg) for 6 weeks. At the end of 2 and 6 weeks rats were injected I.V. immune complex and on the following day the proximal colon was perfused with 2.5% formaldehyde (local irritant 3 ml/hour for 5 mins). Extravasation was measured by Evans' blue technique and neutrophil concentration in the tissue was determined by measuring myeloperoxidase (MPO). AZ did not inhibit extravasation and MPO after 2 weeks of therapy. However, after 6 weeks, AZ reduced extravasation to 20±2 g/gm compared to untreated animals (51±6 g/gm tissue) and MPO levels to 0.3±13 compared to untreated rats (0.8±0.32 mU/gm). There was a good correlation between extravasation and MPO levels. These results suggest that long-term treatment with AZ may prevent extravasation and cause reduction in neutrophil trafficking. Such an effect may be beneficial for maintaining remission in IBD.     

Differential role of neutrophils and monocytes during subcutaneous plasma extravasation

We examined the behavior of polymorphonuclear leukocytes (PMNs) and monocytes during subcutaneous plasma extravasation in guinea-pigs. Plasma extravasation was induced by intradermal injection of zymosan-activated plasma (ZAP). The degree of extravasation correlated logarithmically with the concentration of injected ZAP, and was composed of PMN-dependent and -independent components. The latter was mediated primarily by histamine. The former accounted for 40-50% of the total plasma extravasation, peaked within 15 min, and then rectilinearly decreased with a half-life between 30 and 40 min. Histological examination of skin at 15 min after ZAP injection demonstrated PMN attachment to the luminal surface of venule endothelial cells, without evidence of PMN extravasation. We next examined whether monocyte infiltration of subcutaneous tissue played a causal role in plasma extravasation. Monocyte-predominant infiltration was initially caused by an intradermal injection of a monocyte-specific chemotactic factor, the S19 ribosomal protein (RP S19) dimer. Monocyte infiltration did not induce plasma extravasation even in guinea-pigs with elevated peripheral blood monocyte levels following administration of a macrophage-colony stimulating factor. A simultaneous injection of prostaglandin E2, a vasodilating agent, with RP S19 dimer also did not induce plasma extravasation. In contrast, a simultaneous injection of RP S19 dimer with ZAP changed the leukocyte infiltration pattern from PMN-predominant to monocyte-predominant, and almost completely suppressed the PMN-dependent component of the ZAP-induced plasma extravasation. The lack of plasma extravasation in the monocyte-predominant pattern was reproduced when a strong monocyte infiltration was induced by an intradermal injection of apoptotic cells. We conclude that leukocyte-induced plasma extravasation is specific for PMN, and is not due to a physical leakage of plasma during leukocyte emigration. Rather, plasma extravasation is probably caused by a cognate interaction between PMNs and postcapillary venule endothelial cells.     

Inhibition of Neutrophil‐Dependent Cytotoxicity for Human Endothelial Cells by ACE Inhibitors

Angiotensin‐converting enzyme inhibitors (ACEi) have immunomodulating properties and have been suggested to protect against endothelial injury, for example myocardial infarction and reperfusion injury. We tested whether two ACEi (captopril and enalapril), differing in a thiol group, protected human umbilical vein endothelial cells (HUVEC) from cytotoxicity induced by polymorphonuclear neutrophils (PMN) in vitro, when cells were activated by tumour necrosis factor‐α (TNFα) or the arachidonate derivative lipoxin‐A₄ (LXA₄), using separate cytotoxicity pathways. When ⁵¹Cr labelled HUVEC were treated with captopril (0–500 μm ) or enalapril (0–100 μm ) for 2 h and then activated by TNFα (100 ng/ml) for 24 h, a significant, dose‐dependent reduction of ⁵¹Cr release was observed. Similarly, captopril reduced ⁵¹Cr release when LXA₄ (0.1 μm ) was used to stimulate PMN for 4 h. Among previously defined mechanisms of significance for the cytotoxic reaction, expression of ICAM‐1, but not intracellular Ca²⁺ changes in PMN or PMN adherence to HUVEC, were reduced by ACEi treatment. Moreover, both ACEi inhibited HUVEC surface expression of TNFα receptor I (but not II). Thus, these ACEi, particularly captopril, interfere with PMN‐induced cytotoxicity for endothelial cells by modulating pro‐inflammatory surface receptors, which is a novel effect that might be explored for further therapeutic approaches.     

The Effects of IB4, a Monoclonal Antibody to the CD18 Leukocyte Integrin on Phorbol Myristate Acetate (PMA)-Induced Polymorphonuclear Leukocyte (PMN) Accumulation and Endothelial Injury in Rabbit Lungs

A model of acute lung injury induced by intravenous phorbol myristate acetate (PMA) is described. The model is characterized by the accumulation of polymorphonuclear leukocytes (PMNs) and a hemorrhagic edema in bronchoalveolar lavage (BAL) fluid when measured 6 h following the administration of PMA (60 g/kg, i.v.). It was also determined that PMA induces acute leukopenia and neutropenia which were maximal at 5 min following the injection of PMA and were sustained for at least 6 h, with circulating leukocyte numbers returning to control values by 24 h. The extents to which the inflammatory and systemic changes induced by PMA were dependent on the surface expression on leukocytes of the 2-integrins was assessed by comparing responses to PMA in control animals and animals pretreated with the anti-CD18 monoclonal antibody IB4. The administration of IB4 (1 mg/kg, i.v.) 15 min before PMA did not alter the time course or extent of PMA-induced leukopenia and neutropenia. In contrast IB4 administration (0.1 to 1 mg/kg) produced a dose dependent inhibition of PMN accumulation and plasma extravasation measured in BAL fluid. IB4 (1 mg/kg) completely inhibited PMA evoked increases in plasma extravasation (94.5 ± 1.7%, N = 4) and hemorrhage (95.2 ± 2.1%, N = 4) whereas PMN accumulation in BAL fluid was inhibited by 77.8 ± 3.8% (mean ± SEM, N = 4). Thus, a small, but reproducible, component of the PMA-induced PMN accumulation was not inhibited using this regimen of IB4 administration. If IB4 administration was delayed for 3 h post injection of PMA and bronchoalveolar lavage performed 3 h later, the extents of PMN accumulation and edema formation were similar to those observed 3 h following PMA challenge in control animals not dosed with IB4. This suggests that administration of IB4 during an ongoing inflammatory response is capable of preventing the further development of inflammatory changes and further supports the therapeutic potential of CD18 blockade in conditions such as adult respiratory distress syndrome.     

Simultaneous measurements of the accumulation of isotope-labelled protein and erythrocytes in skin reactions of allergic inflammation in the guinea-pig

This paper describes a method for simultaneous measurement of the accumulation of plasma protein and erythrocytes in skin reactions of hypersensitivity to bovine γ-globulin (BGG) and tuberculin PPD in the guinea-pig. The procedure consists in giving ¹²⁵I-labelled plasma albumin and ⁵¹Cr-labelled autologous erythrocytes together by intravenous injection into guinea-pigs bearing skin lesions of allergic inflammation at different times and for different periods during the development of the skin reactions. Isotope accumulation in excised skin reactions is measured by scintillation counting at different stages during the evolution of hypersensitivity responses.

Skin reactions of combined anaphylactic and Arthus hypersensitivity to BGG were characterized by pronounced increased vascular permeability principally in the first hour. In established 24-hour hypersensitivity reactions to both antigens (BGG and PPD) there was continuing accumulation of both plasma albumin and erythrocytes. During the development of the tuberculin reaction, an intermediate phase of isotope accumulation occurred between 6 and 9 hours after skin testing; serum transfer studies showed that this intermediate peak was not attributable to circulating antibody alone. These isotope tracer techniques were also applied to study vascular permeability in systemically transferred reactions of delayed hypersensitivity and in the vascular response to the intradermal injection of an inflammatory factor generated by antigen-activation of sensitized lymphocytes.

It was concluded that isotope tracing provided objective and sensitive methods for analysing microvascular responses in allergic inflammation.

     

Neutrophil migration,oxidative metabolism,and adhesion in elderly and young subjects

Objective. To evaluate neutrophil functions in the elderly.Methods. We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senn's skin window technique) of 25 normal elderly and of 25 normal young control subjects.Results. No difference was found in PMN migration in vivo (62.9±21.3×10⁶ and 65.5±9.1×10⁶ PMN/cm²/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6±2.7 and 9.3±3.3 nMOLES O ₂ ^– /10⁶ PMN respectively, p<0.0001) and by exudate PMNs (13.6±4.3 and 19.4±6 nMOLES O _2– ^{10 6} PMNs respectively, p<0.005).Conclusion. Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.     

Alterations in complement-induced shape change and stimulus-specific superoxide anion generation by neonatal calf neutrophils

Increased susceptibility of neonates to infection may be related to defects in newborn neutrophil (PMN) functional activities, including altered responses to complement fragments (Cf) and defective microbicidal activity. We therefore compared the kinetics of newborn and adult bovine PMN membrane shape change responses following stimulation with zymosan-activated plasma (ZAP) as a source of Cf. Measurement of PMN membrane shape change was a rapid, sensitive, and reproducible measure of Cf stimulation within a population of PMNs; a maximum of 67–85% of the PMNs exhibited easily detectable membrane ruffling, lamellipodia formation, and polarity within 2 min. Newborn PMNs exhibited significantly increased (P<0.01) membrane shape change at 20, 30, 60, 120, and 300 sec after Cf stimulation. A maximum of 85.8±3.2% of newborn PMNs exhibited such C-finduced shape changes by 120 sec, which was significantly greater (P<0.01) than the maximum stimulation (67.7±4.3%) attained with adult PMNs. These data indicate enhanced kinetics of induced newborn PMN membrane shape change in response to Cf stimulation. We also compared stimulus-specific superoxide anion (O ₂ ^– ) generation as a measure of respiratory burst activity after incubation of new-born and adult PMNs with soluble (phorbol myristate acetate, PMA) and particulate (opsonized zymosan, OZ) stimuli. When PMA was used as the stimulus, newborn PMNs generated significantly less O ₂ ^– (9.3±0.5 nmol O ₂ ^– /10⁶ PMN,P<0.05) than did adult PMNs (12.4±0.3 nmol O ₂ ^– )/10⁶ PMN). This finding was reversed when OZ was used as the stimulus; newborn PMNs generated significantly more O ₂ ^– (7.7±0.4 nmol O ₂ ^– /10⁶ PMN,P <0.05) than did adult PMN (5.5 ±0.5 nmol O ₂ ^– /10⁶ PMN). These findings collectively document biochemical and morphological differences between newborn and adult PMNs as determined by stimulusspecific O ₂ ^– generation and C_f-induced membrane shape change. Such differences may be important to neonatal disease susceptibility.     

Relationship between increased vascular permeability and extravascular albumin clearance in rabbit inflammatory responses induced with Escherichia coli

Intradermal injections of killed Escherichia coli are known to cause a variety of pathophysiological changes in the microcirculation that facilitate the extravasation of plasma constituents into the interstitium. In an attempt to learn more of the factors that regulate the magnitude and duration of inflammatory edema, we have focused on the relationship between the extravasation of protein into the interstitium and the removal of extravascular protein from the lesion sites. Vascular permeability changes have been assessed by the local accumulation of systemically administered [131I] or [125I]-albumin and extravascular protein clearance measured by monitoring the disappearance of [125I]-albumin from the same sites. Radioactivity was quantitated with an external gamma-scintillation probe or by punching out the lesion sites in sacrificed animals and counting in a gamma-spectrometer. Scintillation probe measurements of the net accumulation of intravenously administered [125I]-albumin in E. coli-induced skin lesions revealed that the extravasation of albumin was greater than the clearance of protein from the same sites. Comparisons of the removal rates of albumin injected directly into the E. coli sites revealed that, despite increases in vascular permeability amounting to 170 to 700% of control values, the mobilization of deposited albumin was no greater than that from control tissues that received saline; in fact with high concentrations of E. coli (10(8) injected/site) the mobilization of protein from the lesions was significantly reduced. The systemic administration of 055:B5 endotoxin (0.3, 1.6, or 3.3 micrograms/kg) also suppressed the clearance of albumin from skin. In contrast to these results, 300 to 1500% increases in vascular permeability induced with other inflammatory stimuli including thermal injury, high concentrations of bovine serum albumin, or bradykinin, resulted in enhanced clearance of extravascular protein from lesion or injection sites. These experiments suggest that an inability to effectively mobilize extravascular protein from the inflammatory focus could be a major contributing factor in regulating edema in inflammatory reactions induced with E. coli and may possibly contribute to the edema associated with septicemia.     

PGE1 induced transcapillary transport of 51Cr‐EDTA in rat skin measured by microdialysis

Interstitial fluid pressure (P_if) is a key determinant in increasing the transcapillary driving pressure, pulling fluid from the microcirculation into the interstitial space at the onset of acute inflammatory reactions and the oedema formation associated with these. Prostaglandin E₁ (PGE₁) induces lowering of P_if in rat skin which increases transcapillary transport of ⁵¹Cr‐EDTA into the center of a tumor as measured by microdialysis. The aim of this study was twofold: First, to evaluate and develop the microdialysis technique thoroughly with regard to its suitability for investigating transcapillary water transport in rat skin using ⁵¹Cr‐EDTA as a tracer. Secondly, to evaluate the effect of PGE₁ on transcapillary transport of ⁵¹Cr‐EDTA. This study demonstrates that PGE₁ increases transcapillary transport of ⁵¹Cr‐EDTA into skin interstitium. There were no significant differences between the experimental probe and the control probe when calculations from the entire experiment (90 min) were compared. On the other hand, significant differences were observed by examining the experiment in smaller time intervals. PGE₁ increased transcapillary transport of ⁵¹Cr‐EDTA during the first 15 min when administered through the microdialysis probe. This observation suggests that increased blood flow and/or permeability‐surface area product are responsible for raising the transcapillary transport of ⁵¹Cr‐EDTA, i.e. the transport is diffusion limited. Administration of PGE₁ through the probe rather than around the probe resulted in less scatter between experiments than when PGE₁ was injected subcutaneously around the probe.     

Haemolysis due to formaldehyde-induced anti-N-like antibodies in haemodialysis patients

Summary During reuse of formaldehyde sterilized Kiil-dialysers, red cell survival, measured by means of⁵¹Cr t/2, was significantly reduced (p<0.001) in 16 patients with anti-N-like positive sera, when compared with 19 antibody negative control patients (Mean±SD: 16.5±2.7 versus 22.4±3.1 days.) In antibody negative patients (n=10) replacement of form-aldehyde sterilized dialysers by ethylene-oxide sterilized disposable dialysers resulted in a significant increase (p<0.002) of⁵¹Cr t/2 (Mean±SD, days: Kiil-dialyser 16.3±1.9; disposable dialyser 20.3±3.5). This improvement took place, although antibody titres persisted during the⁵¹Cr-measurements and declined thereafter only slowly. In antibody negative patients (n=6) red cell survival did not increase, when formaldehyde as a sterilant was avoided. In antibody positive patients mean haematocrit rose significantly (p<0.05), whereas in none of the antibody negative patients a definite change of haematocrit occurred. The data demonstrate, that formaldehyde sterilisation of dialysers may cause antibody-mediated haemolysis contributing to the extent of renal anaemia. This immunohaemolysis may be corrected, in spite of continuing antibody persistance, when formaldehyde exposure is totally avoided, or possibly when minimized.     

Quantitation of cutaneous inflammation induced by reactive species generated by UV-visible irradiation of Rose bengal

The present studies were undertaken to quantitate the initial inflammatory response produced by the photo-generated reactive species in rabbit skin. Rose bengal (RB), a photosensensitizer dye, was injected into the skin sites at various concentrations and exposed to UV-visible light for 30–120 min. The increase in vascular permeability and the accumulation of PMNs were investigated using¹²⁵I-labeled albumin and⁵¹Cr-labeled PMNs. RB at a concentration of 1 nmol with 120-min exposure to light enhanced vascular permeability by 3.7 times and accumulation of PMNs by 3.3 times. As low as 0.01 nmol of RB produced discernible effects.-Carofene (0.1 nmole) inhibited the inflammatory response by 75–100%, suggesting that the reactive species involved in this response was predominantly singlet oxygen. The increase in vascular permeability was inhibited by 48–70% by 25g of chlorpheniramine maleate. It is therefore suggested that histamine plays a major role in the initial vascular response. The studies demonstrate that this rabbit model is suitable for the quantitation of photoinduced inflammatory response which is not observable by gross anatomic procedures.Part of this work was presented at NATO Advanced Study Institute on Photosencitisation, July 1987, Kingston, Canada. This work was supported by research grants from Natural Sciences and Engineering Research Council, Canada and Ontario Ministry of Labour, Canada.     

Striated muscle calcium-stimulated cysteine protease (calpain-like) activity promotes myeloperoxidase activity with exercise

 An inflammatory response triggered by neutrophil accumulation into muscle tissue is thought to occur with exercise-induced muscle damage. To investigate the relationship between Ca²⁺-stimulated proteolysis (calpain-like activity) and neutrophil accumulation [myeloperoxidase (MPO) activity], cardiac and plantaris muscles from rats (n = 10) completing 1 h exercise (25 m/min) were investigated. Exercise promoted increases (P<0.05) in both calpain-like and MPO activities; ranging from 2.79 to 58.9 U/g wet weight (ww) and 0.03 to 4.88 U/g ww respectively. Pearson’s correlational analysis (r) on calpain-like and MPO activities for cardiac and plantaris muscle data were 0.97 (P<0.001) and 0.68 (P<0.05) respectively, with a combined r of 0.83 (P<0.001) for both muscles across all conditions. To investigate further the extent to which calpain-like activity may promote neutrophil accumulation, another exercise group (n = 5) was pre-injected with the cysteine protease inhibitor, E64c, 1 h before exercise. Administration of E64c lowered calpain-like and MPO activities by 66% and 56% respectively (average from both muscles). From these results it is concluded that a relationship exists between Ca²⁺-stimulated proteolysis and neutrophil accumulation into striated muscle with exercise, and that the calpain system is involved in localizing the neutrophilic response with exercise. Received: 3 February 1997 / Received after revision: 18 August 1997 / Accepted: 2 December 1997     

Specific binding of 1-[3H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl choline (platelet-activating factor,PAF) by human polymorphonuclear neutrophils

1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) is a potent activator of polymorphonuclear neutrophil (PMN) aggregation, exocytosis and chemotaxis. Specific desensitization of PMN to PAF suggests a receptor-mediated interaction. The binding of 1-[³H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (³H-PAF) to human PMN and platelets was analysed and compared. Binding was saturable at 0.6 nM and 0.1 nM for 2×10⁶ PMN and 5×10⁷ platelets, respectively. The time course of binding at 22°C and 37°C for both cell types reached the plateau at 2 min. The averageK _d was 45.0±1.7 nM (mean ±1 SD of 4 experiments) for PMN (27.391±1381 sites for PMN) and 20.1±6.3 nM (4 experiments) for platelets (1577±461 sites for platelets). The Scatchard plot analysis revealed two distinct binding sites both on PMN and platelets: a high affinity binding site and a non-saturable binding site.This work was supported by C.N.R. Rome grant no. 81.00089.04.     

Regional differences in dermal inflammatory reactions

Regional differences in dermal inflammatory reactions in the dorsum of rat trunk were studied in three commonly used inflammatory models, i.e., reverse passive Arthus reaction (RPAR), passive cutaneous anaphylaxis (PCA), and histamine-induced inflammatory (HI) reaction. The RPAR showed an increasing severity from cranial to caudal regions, as measured by water content in the skin lesions. The PCA reaction, as measured by Evans blue leakage was not influenced by regional differences. The HI reaction, as measured by water content and leakage of radioactively labeled human serum albumin ([¹²⁵I]HSA), was significantly smaller in the central regions of the dorsum than in the most cranial and sacral regions. However, no regional differences were observed when the reaction was evaluated by protein-bound Evans blue leakage. A comparison of the three different methods to determine the HI reaction showed a correlation (r=0.70) between measurements of water content and [¹²⁵I]HSA leakage. There was less correlation of these two methods with measurements of Evans blue skin lesion diameter (r=0.31 and 0.56, respectively). In conclusion, regional differences in inflammatory responses, and methodological differences to measure them, may influence the results of commonly used tests like RPAR, PCA and HI reactions. Such differences should be considered when quantitating dermal inflammatory reactions.     

Identification of antigen-specific neutrophils in the tonsils with recurrent acute inflammation

Abstract

The pathogenesis of recurrent acute tonsillitis (Rtn) is to be further investigated. Polymorphonuclear neutrophils (PMN) often associate with the pathogenesis of acute and chronic inflammation. This study aims to identify the antigen-specific PMNs (sPMNs) isolated from the tonsillar tissues with recurrent acute inflammation. In this study, CD66b⁺ PMNs were isolated from surgically removed tonsils (40 tonsils were from 20 Rtn patients; 24 tonsils were from 12 tonsil tumour patients) by flow cytometry cell sorting. sPMNs were identified through immunological approaches. We found that compared with the control tonsil samples (from marginal non-tumour tissues of tonsil cancer), Rtn samples showed higher PMN frequency, higher levels of myeloperoxidase (MPO) and neutrophil elastase (NE), in which positive correlation was detected between the inflammatory scores in the Rtn tissues and PMN counts (r?=?.7352; p?=?.0002), or MPO (r?=?.6565, p?=?.0017), or NE (r?=?.6687, p?=?.0013). Upon exposure to tonsillar tissue protein extracts in the culture, a portion of Rtn PMNs was activated and released inflammatory mediators. A complex of tonsillar tissue-specific IgG and FcγRI was observed on the surface of Rtn PMNs; these PMNs could specifically recognize the Rtn tissue extracts and were designated the tonsillar antigen-specific PMNs (sPMNs). A signal transduction pathway of mitogen-activated protein kinase (MAPK)-nuclear factor of T cell activation (NFAT) was activated in sPMNs after exposure to Rtn tissue extracts. In summary, a fraction of sPMN in the Rtn tonsillar tissues was identified and characterized. The sPMNs can be activated upon exposure to tonsil-specific antigens. These sPMNs may contribute to the Rtn pathogenesis.     

Peripheral blood mononuclear-cell interleukin-2 production,receptor generation and lymphokine-activated cytotoxicity in inflammatory bowel disease

The interleukin-2 pathway is essential for the normal immune response to antigen stimulation; we have examined the possibility that this may underlie abnormal peripheral blood lymphocyte immunoregulatory function that has been observed in patients with inflammatory bowel disease. We studied 11 patients with Crohn's disease and 5 with ulcerative colitis, all with quiescent disease activity. Peripheral blood mononuclear cells were isolated from these patients and from healthy age- and sex-matched controls. Interleukin-2 production after mitogen and phorbol-myristate acetate stimulation was similar in both groups: 381±71 (mean ± SE) U/ml by control cells and 451±70 by patient cells. Interleukin-2 receptor generation was also measured pre- and poststimulation by labeling with anti-Tac antibody. This was 10.45±1 and 69.95±3.85% for control cells and 11.41±1.38 and 60.9±4.25% for patients cells. Finally, we examined the response of these cells to interleukin-2 stimulation by generating cells with direct cytotoxicity to⁵¹Cr-labeled Daudi-cell targets. Control cells caused 59.5±46%⁵¹Cr release, whereas patient cells caused 50.8±5.18% release. None of the above results achieved statistical significance. We conclude that the peripheral blood interleukin-2 pathway is normal in inactive inflammatory bowel disease.