The therapeutic significance of concomitant antitumor immunity

Summary Intravenous injection of 50 g bacterial endotoxin can cause complete regression of an established SA1 sarcoma, but not if the tumor ir growing in mice that are incapable of generating concomitant immunity because they have been made T cell-deficient by thymectomy and -radiation (TXB mice). It also was shown that endotoxin fails to cause complete regression of a tumor that is either too large or too small. Only when administered on day 9 of tumor growth, at the time of peak concomitant immunity, did endotoxin cause the tumor to undergo complete regression. Direct evidence that the antitumor effect of endotoxin is dependent on concomitant immunity consisted in the demonstration that an SA1 sarcoma growing in TXB recipients can be primed for endotoxin-induced regression by IV infusion of splenic T cells from concomitantly immune donors bearing an endotoxin-susceptible 9-day tumor. Surprisingly, the donor T cells that primed the recipient tumor for endotoxin-induced regression were of the Ly-1⁺2^– phenotype, as evidenced by their susceptibility to treatment with anti-Ly-1 antibody and complement, and their complete resistance to treatment with anti-Ly-2 antibody and complement. They were different, therefore, from the T cells that cause the regression of smaller tumors in -irradiated recipients without the aid of endotoxin. It is suggested that the antitumor function of endotoxin depends on its ability to cause intratumor macrophages to acquire and express tumoricidal function, but only after the macrophages have been activated by Ly-1⁺2^– tumor-sensitized T cells.     

Poractant alfa (Curosurf?) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo

Background

Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e.g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf^®) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages.

Methods

Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages.

Results

Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage.

Conclusions

We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.     

Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin 1, endotoxin, and tumor-promoting phorbol diesters

Cultured human vascular endothelial cells obtained from umbilical cord veins were observed to acquire adhesive properties for purified neutrophils after exposure to IL 1, endotoxin, and tumor-promoting phorbol diesters. Adhesiveness induced by IL1 and endotoxin had similar kinetics of onset, producing no change after 30 min incubation and reaching optimal change by 4 hr of incubation. The phorbol diester TPA induced changes in adhesiveness more rapidly, with half maximal increase induced by a 15- to 30-min exposure. TPA, but not IL 1 or LPS, induced significant morphologic changes in the endothelial cell monolayer. None of the stimuli decreased endothelial cell viability. All stimuli induced increased adhesiveness at relevant concentrations, i.e., endotoxin, 0.01 to 1 microgram/ml; IL 1, 0.5 to 2 U/ml; and TPA, 1 to 30 ng/ml. Structure activity relationships among phorbol diesters indicate that the response occurs through a typical phorbol diester "receptor." A protein synthesis inhibitor (cycloheximide) and an RNA synthesis inhibitor (actinomycin D) prevented the acquisition of adhesiveness stimulated by IL 1 and endotoxin but not by TPA. In addition, TPA showed a differential temperature sensitivity in inducing adhesiveness in endothelial cells. IL 1 and endotoxin did not produce the effect with a 4-hr incubation at 22 degrees or 4 degrees C, whereas TPA was effective at these lower temperatures. Purified human IL 2 and recombinant-derived interferon-gamma failed to induce adhesiveness in vascular endothelial cells, indicating that this is not a general property of lymphokines. We conclude that endothelium may, under some circumstances, play an active role in producing a leukocyte infiltrate at a local tissue site by acquiring adhesive properties. The production of IL 1 by tissue macrophages, etc., may serve as an important initiator of an inflammatory cell infiltrate. Finally, an action of tumor-promoting phorbol diesters in increasing endothelial cell adhesiveness, combined with their known effects in activating leukocytes, may help explain the extraordinary inflammatory potency of these compounds.     

Free radicals,cytokines and nitric oxide in cardiac failure and myocardial infarction

Myocardial infarction is the most common cause of congestive cardiac failure. Free radicals, cytokines, nitric oxide (NO) and antioxidants play a major role both in atherosclerosis and myocardial damage and preservation. In the early stages of atherosclerosis, neutrophils and monocytes infiltrate the intima and generate free radicals which damage the endothelial cells. As a result, production of NO and prostacyclin by the endothelial cells declines, which have cardioprotective actions. This also has relevance to the beneficial action of aspirin since, it can modulate both prostanoid and l-arginine-NO systems and NF-kB translocation. In both acute myocardial infarction and chronic congestive cardiac failure, the plasma levels of various inflammatory mediators such as interleukins and tumour necrosis factor- (TNF) are elevated. TNF, produced by the inflammatory cells and the myocardium, can suppress myocardial contractility and induce the production of free radicals, which in turn can further damage the myocardium. Transforming growth factor (TGF), polyunsaturated fatty acids and the glucose-insulin-potassium regimen can antagonize the harmful actions of TNF and protect the myocardium. This explains why efforts made to reduce the levels of pro-inflammatory cytokines have beneficial action and preserve the myocardium.     

Treatment with TNF-α or bacterial lipopolysaccharide attenuates endocardial endothelial cell-mediated stimulation of cardiac fibroblasts

Background

The endocardial endothelium that lines the inner cavity of the heart is distinct from the microvascular endothelial cells and modulates cardiac muscle performance in a manner similar to the vascular endothelial modulation of vascular structure and vasomotor tone. Although the modulatory effects of endocardial endothelium (EE) on cardiomyocytes are firmly established, the regulatory effects of endocardial endothelium on the cardiac interstitium and its cellular components remain ill defined.

Methods and Results

We investigated whether the stimulatory effect of EE on cardiac fibroblasts would be altered when EECs are activated by the cytokine tumor necrosis factor-α (TNF-α) or the endotoxin bacterial lipopolysaccharide (LPS). Both TNF-α and LPS were found to independently attenuate the stimulatory effect of EE on cardiac fibroblasts. These agents lowered the synthesis or release of ET-1 and increased the secretion of TGF-β and NO.

Conclusion

The findings of this study using endocardial endothelial cells (EECs) and neonatal cardiac fibroblasts demonstrate that pro-inflammatory cytokines cause altered secretion of paracrine factors by EECs and inhibit proliferation and lower collagen synthesis in fibroblasts. These changes may influence fibroblast response and extra cellular matrix remodeling in pathological conditions of the heart.     

VISTA deficiency attenuates antibody-induced arthritis and alters macrophage gene expression in response to simulated immune complexes

Background

In addition to activated T cells, the immune checkpoint inhibitor “V domain-containing Ig suppressor of T-cell activation” (VISTA) is expressed by myeloid cell types, including macrophages and neutrophils. The importance of VISTA expression by myeloid cells to antibody-induced arthritis and its potential for relevance in human disease was evaluated.

Methods

VISTA was immunolocalized in normal and arthritic human synovial tissue sections and synovial tissue lysates were subjected to western blot analysis. The collagen antibody-induced arthritis model (CAIA) was performed with DBA/1 J mice treated with antibodies against VISTA and with VISTA-deficient mice (V-KO). Total mRNA from arthritic joints, spleens, and cultured macrophages was analyzed with NanoString arrays. Cytokines secreted by splenic inflammatory macrophages were determined. In-vitro chemotaxis and signal transduction assays were performed with cultured macrophages.

Results

VISTA protein was localized to synovial membrane cells, neutrophils, and scattered cells in lymphocyte-rich foci and was detected by western blot analysis in normal synovium and synovium from rheumatoid arthritis patients. Deficiency of VISTA or treatment of mice with anti-VISTA monoclonal antibodies attenuated CAIA. Joint damage and MMP-3 expression were significantly reduced in V-KO mice. Surface expression of C5a receptor was reduced on monocytes, neutrophils, and cultured macrophages from V-KO. Upon Fc receptor engagement in vitro, gene expression by V-KO macrophages was altered profoundly compared to WT, including a significant induction of IL-1 receptor antagonist (IL1rn).

Conclusions

VISTA expression supports immune-complex inflammation in CAIA and VISTA is expressed in human synovium. VISTA supports optimal responses to C5a and modulates macrophage responses to immune complexes.

     

Differential effects of human blood monocytes on the growth of human tumour cell lines in vitro

Summary Monocytes and macrophages have been shown to be cytotoxic towards tumour cells in vitro. However, although tumour-associated monocytes and macrophages are now widely accepted to contribute a relatively high proportion of the cellular infiltrate of experimental and human solid carcinomas, a cytotoxic/cytostatic effector function for these cells in vitro or in vivo has yet to be conclusively demonstrated. In the present study, we show that non-activated peripheral blood monocytes co-cultured with tumour cells across a semi-permeable membrane release soluble factors that modulate the growth of tumour cells in contrasting ways. After Nycoprep 1.068 separation, non-activated peripheral blood monocytes enhanced the in vitro proliferation of HT29 colon adenocarcinoma cells but inhibited T47D breast carcinoma cell replication; peripheral blood lymphocytes were incapable of mediating these effects. In contrast, peripheral blood monocytes activated by interferon caused a pronounced inhibition of both HT29 and T47D cell proliferation.     

Neutrophils reduce the parasite burden in Leishmania (Leishmania) amazonensis-infected macrophages

Background

Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L) major, whereas less information is available for L. (L) amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L.) amazonensis (C3H/HePas). In contrast, the susceptible strain (BALB/c) displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L.) amazonensis-infected macrophages in vitro.

Methodology/Principal Findings

Mouse peritoneal macrophages infected with L. (L.) amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1) intracellular parasites were efficiently destroyed in the co-cultures; 2) the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas) or susceptible (BALB/c) to L. (L.) amazonensis; 3) parasite destruction did not require contact between infected macrophages and neutrophils; 4) tumor necrosis factor alpha (TNF-α), neutrophil elastase and platelet activating factor (PAF) were involved with the leishmanicidal activity, and 5) destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.

Conclusions/Significance

The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L.) amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.     

Dystroglycan Depletion Impairs Actin-Dependent Functions of Differentiated Kasumi-1 Cells

Background

Dystroglycan has recently been characterised in blood tissue cells, as part of the dystrophin glycoprotein complex involved in the differentiation process of neutrophils.

Purpose

In the present study we have investigated the role of dystroglycan in the human promyelocytic leukemic cell line Kasumi-1 differentiated to macrophage-like cells.

Methods

We characterised the pattern expression and subcellular distribution of dystroglycans in non-differentiated and differentiated Kasumi-1 cells.

Results

Our results demonstrated by WB and flow cytometer assays that during the differentiation process to macrophages, dystroglycans were down-regulated; these results were confirmed with qRT-PCR assays. Additionally, depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated Kasumi-1 cells, including morphology, migration and phagocytic activities although secretion of IL-1β and expression of markers of differentiation are not altered.

Conclusion

Our findings strongly implicate dystroglycan as a key membrane adhesion protein involved in actin-based structures during the differentiation process in Kasumi-1 cells.     

Dietary glycine blunts lung inflammatory cell influx following acute endotoxin

Mortality associated with endotoxin shock is likely mediated by Kupffer cells, alveolar macrophages, and circulating neutrophils. Acute dietary glycine prevents mortality and blunts increases in serum tumor necrosis factor-alpha (TNF-alpha) following endotoxin in rats. Furthermore, acute glycine blunts activation of Kupffer cells, alveolar macrophages, and neutrophils by activating a glycine-gated chloride channel. However, in neuronal tissue, glycine rapidly downregulates chloride channel function. Therefore, the long-term effects of a glycine-containing diet on survival following endotoxin shock were investigated. Dietary glycine for 4 wk improved survival after endotoxin but did not improve liver pathology, decrease serum alanine transaminase, or effect TNF-alpha levels compared with animals fed control diet. Interestingly, dietary glycine largely prevented inflammation and injury in the lung following endotoxin. Surprisingly, Kupffer cells from animals fed glycine for 4 wk were no longer inactivated by glycine in vitro; however, isolated alveolar macrophages and neutrophils from the same animals were sensitive to glycine. These data are consistent with the hypothesis that glycine downregulates chloride channels on Kupffer cells but not on alveolar macrophages or neutrophils. Importantly, glycine diet for 4 wk protected against lung inflammation due to endotoxin. Chronic glycine improves survival by unknown mechanisms, but reduction of lung inflammation is likely involved.     

N-Acetylcysteine inactivates Migration Inhibitory Factor and Delayed Hypersensitivity Reactions

In vitro studies suggest that delayed hypersensitivity follows the production of migration inhibitory factor (MIF) by sensitive lymphocytes in the presence of specific antigen. This factor arrests the migration of macrophages in vitro and in vivo. After attraction, aggregation and activation in vivo, these bystander cells produce toxic substances which induce the local reaction¹. When lymphocytes from tuberculin (PPD) sensitized guinea-pigs were incubated with PPD, cell-free supernatant fluids of the cultures contained MIF². Such migration inhibitory fluids injected intradermally with PPD, into PPD-sensitive animals, enhanced the delayed hypersensitivity reaction³. Concentrated migration inhibitory supernatant fluids injected intradermally into unsensitized animals produced local reactions of induration and erythema within 6 h; reactions reached a maximum after 16 h. Histologically there was an infiltrate of mononuclear cells at the site of injection and neutrophils and eosinophils were also present¹.     

Transmission potential,skin inflammatory response,and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background

Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector.

Results

The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs).

Conclusion

Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

     

Regulation of endothelial cell adhesion molecule expression by mast cells, macrophages, and neutrophils

Background

Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined.

Methods and Results

Using bone marrow-derived mast cells from wild-type, Tnf^−/−, Ifng^−/−, Il6^−/− mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC.

Conclusion

Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases.     

CD4-Positive T Cells and M2 Macrophages Dominate the Peritoneal Infiltrate of Patients with Encapsulating Peritoneal Sclerosis

Background

Encapsulating peritoneal sclerosis (EPS) is a severe complication of peritoneal dialysis (PD). Previously, it has been shown that infiltrating CD4-positive T cells and M2 macrophages are associated with several fibrotic conditions. Therefore, the characteristics of the peritoneal cell infiltrate in EPS may be of interest to understand EPS pathogenesis. In this study, we aim to elucidate the composition of the peritoneal cell infiltrate in EPS patients and relate the findings to clinical outcome.

Study Design, Setting, and Participants

We studied peritoneal membrane biopsies of 23 EPS patients and compared them to biopsies of 15 PD patients without EPS. The cellular infiltrate was characterized by immunohistochemistry to detect T cells(CD3-positive), CD4-positive (CD4+) and CD8-positive T cell subsets, B cells(CD20-positive), granulocytes(CD15-positive), macrophages(CD68-positive), M1(CD80-positive), and M2(CD163-positive) macrophages. Tissues were analysed using digital image analysis. Kaplan-Meier survival analysis was performed to investigate the survival in the different staining groups.

Results

The cellular infiltrate in EPS biopsies was dominated by mononuclear cells. For both CD3 and CD68, the median percentage of area stained was higher in biopsies of EPS as opposed to non-EPS patients (p<0.001). EPS biopsies showed a higher percentage of area stained for CD4 (1.29%(0.61-3.20)) compared to CD8 (0.71%(0.46-1.01), p = 0.04), while in the non-EPS group these cells were almost equally represented (respectively 0.28%(0.05-0.83) versus 0.22%(0.17-0.43), p = 0.97). The percentage of area stained for both CD80 and CD163 was higher in EPS than in non-EPS biopsies (p<0.001), with CD163+ cells being the most abundant phenotype. Virtually no CD20-positive and CD15-positive cells were present in biopsies of a subgroup of EPS patients. No relation was found between the composition of the mononuclear cell infiltrate and clinical outcome.

Conclusions

A characteristic mononuclear cell infiltrate consisting of CD4+ and CD163+ cells dominates the peritoneum of EPS patients. These findings suggest a role for both CD4+ T cells and M2 macrophages in the pathogenesis of EPS.     

Effect of erythrocyte transfusion on the circulation of Guérin carcinoma-bearing rats

In Guérin carcinoma-bearing rats during tumour growth cardiac output and blood flow to several organs were increased whereas TPR, vascular resistance of some regions and haematocrit gradually decreased. In response to erythrocyte transfusion the anaemia of tumour-bearing rats was considerably improved or corrected, however, the parameters for systemic and regional circulation were only partially restored. We presume that the circulatory "hyperkinesis" of tumour-bearing rats is partly due to the anaemia but also an unknown factor must be considered. The blood perfusion of the Guérin carcinoma was not affected by the erythrocyte transfusion thus it is feasible that the tumour vasculature is maximally dilated even with a normal haematocrit of the host.     

Tumour cell apoptosis modulates the colorectal cancer immune microenvironment via interleukin-8-dependent neutrophil recruitment

Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66b^highCD11b^highCD62L^low phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86^lowCD163⁺CD206⁺ phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis.Subject terms: Cancer microenvironment, Cell death and immune response     

Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock

Effects of endotoxin administration on the ATP-dependent Ca²⁺ uptake by canine cardiac sarcoplasmic reticulum (SR) were investigated. Results obtained 4 h after endotoxin administration show that ATP-dependent Ca²⁺ uptake by cardiac SR was decreased by 27–43% (p < 0.05). Kinetic analysis indicates that the Vmax values for Ca²⁺ and for ATP were significantly decreased while the S_0.5 and the Hill coefficient values were not affected during endotoxin shock. Magnesium (1–5 mM) stimulated while vanadate (25–50 M) inhibited the ATP-dependent Ca²⁺ uptake, but the Mg²⁺-stimulated and the vanadate-inhibited activities remained significantly lower in the endotoxin-treated animals. Phosphorylation of SR by the exogenously added catalytic subunit of the cAMP-dependent protein kinase or by the addition of calmodulin stimulated the ATP-dependent Ca²⁺ uptake activities both in the control and endotoxin-injected dogs. However, the phosphorylation-stimulated activities remained significantly lower in the endotoxin-injected dogs. Dephosphorylation of SR decreased the ATP-dependent Ca²⁺ uptake, but the half-time required for the maximal dephosphorylation was reduced by 31% (p < 0.05) 4 h post-endotoxin. These data indicate that endotoxin administration impairs the ATP-dependent Ca²⁺ uptake in canine cardiac SR and the endotoxininduced impairment in the SR calcium transport is associated with a mechanism involving a defective phosphorylation and an accelerated dephosphorylation of SR membrane protein. Since ATP-dependent Ca²⁺ uptake by cardiac SR plays an important role in the regulation of the homeostatic levels of the contractile calcium, our findings may provide a biochemical explanation for myocardial dysfunction that occurs during endotoxin shock.     

Microscopical study of experimental wound healing in <Emphasis Type="Italic">Notothenia coriiceps</Emphasis> (Cabeçuda) at 0°C

Notothenia coriiceps (Cabeçuda) is an Antarctic benthic fish frequently found with lesions in the tegument caused by seal predation. We have investigated epidermal repair in these animals by means of a microscopic study of experimental wound healing at 0°C. At 24–48 h after wound induction, mucous exudate and necrotic lining cells covered the wound. At 7–14 days, an epidermal tongue could be discerned, folded at the tip, with intercellular oedema between the tip and the wound border. After 23–30 days, the wound was completely closed and the migrating epidermis, with intercellular oedema, was reduced. By 45–90 days, melanocytes progressively increased in the epidermis but no scales were formed. The inflammatory infiltrate was mainly composed of neutrophils after 7 days, at which time they were mostly replaced by macrophages; lymphocytes and plasma cells were also present. The border epidermis slid towards the centre, folding at the tip and finally fusing to form a diaphragm. The cells of the epidermis began to multiply only after complete closure of the wound. The lack of scale formation on induced and naturally found wounds, even after 90 days, suggests that different mechanisms in wound repair occur at 0°C from those in fish from temperate and tropical environment. This is the first report of successful wound repair at polar temperatures, indicating the adaptation of N. coriiceps to the Antarctic environment.The financial support from CNPq (68.0047/00-0 and 48.0262/00-4 grants), PROANTAR, SECIRM and FAPESP is greatly appreciated. Professor E.L. Cooper is partially supported by funds from the Hewlett Foundation to the Latin American Center, UCLA.     

Distinct synovial immunopathology in Behçet disease and psoriatic arthritis

Introduction

The aim of the study was to investigate synovial immunopathology differences between early Behçet disease (BD) and psoriatic arthritis (PsA).

Methods

Needle arthroscopy of an inflamed knee joint was performed in patients with early untreated BD (n = 8) and PsA (n = 9). Synovial fluid (SF) was collected for cytokines, perforin, and granzyme analysis. Eight synovial biopsies per patient were obtained for immunohistochemical analysis of the cellular infiltrate (T cells, natural killer cells, macrophages, B cells, plasma cells, mast cells, and neutrophils), blood vessels as well as expression of perforin and granzyme. The stained slides were evaluated by digital image analysis.

Results

The global degree of synovial inflammation was similar in the two types of arthritis. In the analysis of the innate immune cell infiltration, there was a striking neutrophilic inflammation in BD synovitis whereas PsA displayed significantly higher numbers of cells positive for c-kit, a marker of mast cells. As for lymphocytes, CD3⁺ T cells, but neither CD20⁺ B cells nor CD138⁺ plasma cells, were significantly increased in BD versus PsA. Further analysis of the T-lymphocyte population showed no clear shift in CD4/CD8 ratio or Th1/Th2/Th17 profile. The SF levels of perforin, an effector molecule of cytotoxic cells, displayed a significant four- to fivefold increase in BD.

Conclusions

This systematic comparative analysis of early untreated synovitis identifies neutrophils and T lymphocytes as important infiltrating cell populations in BD. Increased levels of perforin in BD suggest the relevance of cytotoxicity in this disease.