Influence of cytokines on matrix metalloproteinases produced by fibroblasts cultured in monolayer and collagen gels.

BACKGROUND: Extracellular matrix metalloproteinases (MMPs) are crucial factors involved in connective tissue remodeling that accompanies ultraviolet radiation-induced actinic damage. This study investigated whether the cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-10 modulate the expression of MMPs in cultured human newborn skin fibroblasts. METHODS: Different concentrations of TNF-alpha, IL-1 beta, and IL-10 were added to human dermal fibroblasts grown in monolayers or embedded in three-dimensional (3D) collagen gels, a model closer to skin. Supernatant from the fibroblast cell culture was collected 24 hours later. The concentrations of MMP-1 and MMP-3 were assaysed by enzyme-linked immunosorbent assay (ELISA) while the concentrations of MMP-2 and MMP-9 were analysed by zymography. RESULTS: Basal production of MMPs was significantly greater in fibroblasts grown in 3D gels than in cells grown as monolayers. TNF-alpha and IL-1 beta induced increases in the concentrations of MMP-1, MMP-3, and MMP-9, but not in MMP-2 or tissue inhibitor of matrix metalloproteinase (TIMP)-1 or -2. The inducibility of MMP secretion is more significant in 3D gels. IL-10 did not significantly modulate MMPs. CONCLUSION: This study demonstrated that basal concentrations of MMPs are higher in fibroblasts cultured in 3D gels and their response to cytokines is different to that of cells grown as monolayers. Cytokines can increase the collagenolytic and gelatinolytic activity involved in extracellular matrix remodeling and hence contribute to photoaging.

Laser-induced shock waves enhance sterilization of infected vascular prosthetic grafts.

BACKGROUND AND OBJECTIVE: Bacteria that cause infection of vascular prosthetic grafts produce an exopolysaccharide matrix known as biofilm. Growth in biofilms protects the bacteria from leukocytes, antibodies and antimicrobial drugs. Laser-generated shock waves (SW) can disrupt biofilms and increase drug penetration. This study investigates the possibility of increasing antibiotic delivery and sterilization of vascular prosthetic graft. STUDY DESIGN/MATERIALS AND METHODS: Strains of Staphylococcus epidermidis and S. aureus were isolated from infected prosthetic grafts obtained directly from patients. Dacron grafts were inoculated with the isolated bacteria, which were allowed to form adherent bacterial colonies. The colonized grafts underwent the following treatments: (a) antibiotic (vancomycin) alone; (b) antibiotic and SW (c) saline only; and (d) saline and SW. Six hours after treatment, the grafts were sonicated, the effluent was cultured and the colony forming units (CFU) were counted. RESULTS: CFU recovered from control grafts colonized by S. epidermidis were comparable: saline, 3.05 x 10(8) and saline+SW 3.31 x 10(8). The number of S. epidermidis CFU diminished to 7.61 x 10(6) after antibiotic treatment but the combined antibiotic+SW treatment synergistically decreased CFU number to 1.27 x 10(4) (P<0.001). S. aureus showed a higher susceptibility to the antibiotic: 2.26 x 10(6) CFU; antibiotic +SW treatment also had an incremental effect: 8.27 x 10(4) CFU (P<0.001). CONCLUSIONS: This study demonstrates that laser-generated shock waves have no effects alone, but can enhance the effectiveness of antibiotics against bacteria associated with prosthetic vascular graft biofilms, suggesting that this treatment may be of value as adjunctive therapy for prosthetic graft infections.

Clinical potential of photodynamic therapy in cardiovascular disorders.

Atherosclerosis and intimal hyperplasia remain obstacles for surgeons to overcome following vascular reconstructions. Even with all of the technical improvements that have occurred in the past several decades, long term patency following intervention is hindered by these inherent adverse developments. Today, the use of light is seen as a potential treatment modality in vascular surgery. Photodynamic therapy (PDT) has been used in the treatment of cancer, and because of its continued success in vascular experimental models it is now being tested in clinical trials for vascular diseases. PDT offers the surgeon many advantages, and it may have unlimited uses in the clinical setting. Is PDT the ultimate treatment modality for the cardiovascular surgeon and will it help to overcome the inherent failures associated with vascular reconstructions? It may be too early to answer these questions, but with the current successes demonstrated by PDT, there is a need for further testing in clinical trials. In the near future, PDT may be used clinically as a treatment modality to inhibit restenosis and intimal hyperplasia following surgical intervention.

Photodynamic therapy induces apoptosis in intimal hyperplastic arteries.

Photodynamic therapy (PDT) generates free radicals through the absorption of light by photosensitizers. PDT shows promise in the treatment of intimal hyperplasia, which contributes to restenosis, by completely eradicating cells in the vessel wall. This study investigates the mechanisms of PDT-induced cell death. PDT, using the photosensitizer chloroaluminum-sulfonated phthalocyanine (1 mg/kg) and laser light (lambda = 675 nm) 100 J/cm(2) was administered to rat carotid arteries after balloon injury-induced intimal hyperplasia. Apoptosis was determined by cell morphology with light microscopy and transmission electron microscopy, DNA cleavage by terminal dUTP nick-end labeling staining, and nucleosomal fragmentation (ladder pattern) by DNA agarose gel electrophoresis. Four hours after PDT, apoptosis was observed in vascular cells, as evidenced by terminal dUTP nick-end labeling staining and transmission electron microscopy. Within 24 hours no cells were present in the neointima and media. Immunofluorescence using an alpha-smooth muscle cell actin antibody confirmed the disappearance of all neointimal and medial cells within 24 hours. No inflammatory cell infiltrate was observed during this time frame. Apoptosis was sharply confined to the PDT treatment field. These data demonstrate that vascular PDT induces apoptosis as a mechanism of rapid, complete, and precise cell eradication in the artery wall. These findings and the lack of inflammatory reaction provide the basis for understanding and developing PDT for a successful clinical application in the treatment of hyperplastic conditions such as restenosis.

Photodynamic therapy generates a matrix barrier to invasive vascular cell migration.

Photodynamic therapy (PDT) inhibits experimental intimal hyperplasia. PDT results in complete vascular wall cell eradication with subsequent adventitia but minimal media repopulation. This study was designed to test the hypothesis that PDT alters the vascular wall matrix thereby inhibiting invasive cell migration, and as such, provides an important barrier mechanism to favorably alter the vascular injury response. Untreated smooth muscle cells (SMCs) and fibroblasts were seeded on control and PDT-treated (100 J/cm(2); photosensitizer was chloroaluminum-sulfonated phthalocyanine, 5 microg/mL) 3-dimensional collagen matrix gels. Invasive cell migration was temporally quantified by calibrated microscopy. Zymography and ELISA assessed SMC matrix metalloproteinase levels. Molecular changes of gel proteins and their susceptibility to collagenase were analyzed by SDS-PAGE and Western blot. Limited pepsin digestion and histology were used to assess the in vivo relevance of the model, using an established rat carotid artery model at 1 and 4 weeks after balloon injury and PDT. PDT of 3-dimensional matrix of gels led to a 52% reduction of invasive SMCs and to a 59% reduction of fibroblast migration (P<0.001) but did not significantly affect secretion of matrix metalloproteinases. PDT induced collagen matrix changes, including cross-linking, which resulted in resistance to protease digestion. PDT led to a durable 45% reduction in pepsin digestion susceptibility of treated arteries (P<0.001) and inhibition of periadventitial cell migration into the media. These data suggest that PDT of matrix gels generates a barrier to invasive cellular migration. This newly identified effect on matrix proteins underscores its pleiotropic actions on the vessel wall, and as such, PDT may be of considerable potential therapeutic value to inhibit restenosis.

Human neutrophils secrete gelatinase B in vitro and in vivo in response to endotoxin and proinflammatory mediators.

Bacterial sepsis is characterized by a systemic inflammatory state, with activation of numerous cell types. Phagocytes participate in this phenomenon by secreting various proinflammatory cytokines and enzymes. Matrix metalloproteinases (MMPs) such as gelatinases are produced by phagocytes and are thought to play an important role in processes of cell transmigration and tissue remodeling. In this work, we show that endotoxin (lipopolysaccharide [LPS]) and other inflammatory mediators, such as tumor necrosis factor (TNF), interleukin-8, and granulocyte colony-stimulating factor, induce a rapid (within 20 min) release of gelatinase-B (MMP-9) zymogen in whole human blood, as determined by gelatin zymography. The polymorphonuclear neutrophil was identified as the cell responsible for this rapid secretion, as a result of the release of preformed enzymes stored in granules. Normal human subjects given LPS intravenously showed a similar pattern of proMMP-9 secretion, with maximum plasma levels reached 1.5 to 3 h after LPS administration (P = 0.0009). Prior administration of TNF receptor:Fc, a potent TNF antagonist, to subjects given LPS, only partially blunted the release of proMMP-9 (P = 0.033). Ibuprofen, a cyclooxygenase inhibitor, did not alter this pattern of release. Increased levels of proMMP-9 and proMMP-2, as well as activated forms of MMP-9, were found in plasma from two patients with gram-negative sepsis. The levels of MMPs paralleled the severity of clinical condition and a marker of the severity of sepsis, plasma procalcitonin. These data indicate that MMPs are released in whole blood in response to various inflammatory mediators and that they could serve as sensitive and early markers for cell activation during the course of bacterial sepsis.

An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung.

Microvascular endothelial cells (MVEC), which differ from large vessel endothelial cells, have been isolated successfully from lungs of various species, including man. However, contamination by nonendothelial cells remains a major problem in spite of several technical improvements. In view of the organ specificity of MVEC, endothelial cells should be derived from the tissue involved in the diseases one wishes to study. Therefore, to investigate some of the immunopathological mechanisms leading to acute respiratory distress syndrome (ARDS), we have attempted to isolate lung MVEC from patients undergoing thoracic surgery for lung carcinoma and patients dying of ARDS. The method described here includes four main steps: (1) full digestion of pulmonary tissue with trypsin and collagenase, (2) aggregation of MVEC induced by human plasma, (3) Percoll density centrifugation, and (4) selection and transfer of MVEC after local digestion with trypsin/EDTA under light microscopy. Normal and ARDS-derived lung MVEC purified by this technique presented contact inhibition (i.e., grew in monolayer), and expressed classical endothelial markers, including von Willebrand factor (vWF), platelet endothelial cell adhesion molecule 1(PECAM-1, CD31), and transcripts for the angiotensin converting enzyme (ACE). The cells also formed capillarylike structures, took up high levels of acetylated low-density lipoprotein (Ac-LDL), and exhibited ELAM-1 inducibility in response to TNF. Contaminant cells, such as fibroblasts, smooth muscle cells, or pericytes, were easily recognized on the basis of morphology and were eliminated by selection of plasma-aggregated cells under light microscopy. The technique presented here allows one to study the specific involvement and contribution of pulmonary endothelium in various lung diseases.

Interleukin-10 modulates susceptibility in experimental cerebral malaria.

In this study, we examined the effects of interleukin-10 (IL-10) on the outcome of experimental cerebral malaria (CM), a lethal neurological syndrome that occurs in susceptible strains of mice after infection with Plasmodium berghei ANKA (PbA). Constitutive IL-10 mRNA levels were significantly higher in the spleen and brain of resistant animals. In vivo neutralization of endogenous IL-10 in CM-resistant mice induced the neurological syndrome in 35.7% of these mice, as opposed to 7.7% in controls. IL-10 inhibited PbA antigen-specific interferon-gamma (IFN-gamma) production in vitro but not tumour necrosis factor (TNF) serum levels in vivo. Susceptible mice, on the other hand, were significantly protected against CM when injected with recombinant IL-10. Overall, our findings suggest that IL-10 plays a protective role against experimental cerebral malaria.

Changes in endogenous cytokines, adhesion molecules and platelets during cytokine-induced tumour necrosis.

The aim of this study was to investigate mechanisms of anti-tumour activity and necrosis induced by combinations of tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma). In a breast cancer xenograft model, locally injected recombinant human TNF-alpha arrested growth of established tumours in the absence of overt necrosis. Macroscopic necrosis occurred when rat IFN-gamma, which had no anti-tumour activity as a single agent, was given systemically. Treatment with TNF-alpha and IFN-gamma caused focal engorgement of tumour capillaries with erythrocytes, intravascular recruitment of polymorphonuclear cells and platelet adherence to the tumour vascular endothelium 4 h after the combined treatment. This was followed by destruction of tumour vascular endothelium and both necrosis and apoptosis of tumour cells. Concomitant with these changes, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the increase of stromal (murine) mRNA levels for TNF-alpha, TNF receptor 55 kDa, TNF receptor 75 kDa, intracellular adhesion molecule 1, vascular cell adhesion molecule 1, P-selectin and interleukin 6 (IL-6). Thus, the effect of the combined TNF-alpha and IFN-gamma therapy involved the selective destruction of the tumour vasculature, death of tumour cells and increased expression of a series of stromal cytokines, cytokine receptors and adhesion molecules, which could be implicated in the observed events.

Assaying tumor necrosis factor concentrations in human serum. A WHO International Collaborative study.

A collaborative study involving several international research groups was conducted in order to test the validity and reproducibility of tumor necrosis factor-alpha (TNF) measurements in serum. 58 serum samples, nine of them spiked with recombinant human TNF, were aliquoted and distributed blindly to 11 different laboratories. 20 samples were obtained from cerebral malaria patients, 20 from septic shock patients, eight from patients with rheumatoid arthritis and ten from normal blood donors. The serum samples were assayed for TNF using various immunoassays (ELISA), radioimmunoassays (RIA) and bioassays. Interassay coefficient of variance was analysed. Substantial differences were observed on terms of sensitivity and results obtained with the different commercial and in-house assays. The level of sensitivity was highest with ELISAs and bioassays. RIAs yielded the highest concentrations of TNF in the same samples as compared to those obtained by ELISAs and bioassays. These data emphasize the necessity of establishing international standards for cytokine assays in order to render the interpretation of biological and medical data more reliable.

Expression of the tumor necrosis factor gene by dermal fibroblasts in response to ultraviolet irradiation or lipopolysaccharide.

To examine the effects of different wavelengths of ultraviolet (UV) radiation on tumor necrosis factor (TNF) production, we took advantage of mice carrying a chloramphenicol acetyl transferase (CAT) reporter transgene bearing the entire TNF promoter and 3'-untranslated region. Aside from constitutive expression in the thymus, CAT activity was detected only in locally UVB- or UVC-irradiated skin. After UVB irradiation, markedly greater amounts of CAT activity were traced to the dermis rather than the epidermis; by contrast, almost all CAT activity was localized to the epidermis after UVC irradiation. Fibroblasts have not been shown previously to express the TNF gene, i.e., the TNF gene is highly methylated and inaccessible to exogenous modulation in 3T3 fibroblasts. However, the present report reveals that cultured dermal fibroblasts are capable of producing both CAT and TNF in response to treatment in vitro with either UVB irradiation, UVC irradiation, or lipopolysaccharide. These findings indicate that dermal fibroblasts may serve not only as a target for but also as a source of TNF.

Tumor necrosis factor alpha (TNF-alpha) and TNF-beta and their receptors in experimental cutaneous leishmaniasis.

Experimental infection of BALB/c mice with Leishmania major leads to lesions which progress without healing and visceralization, reproducing the most severe forms of human leishmaniasis, while resistant mice like CBA spontaneously resolve lesions and develop protective immunity. Given the conflicting data pertaining to the role of tumor necrosis factor alpha (TNF) in Leishmania infection, we analyzed the expression of TNF, tumor necrosis factor beta (lymphotoxin), and TNF receptor type I (TNF-RI) and type II (TNF-RII) genes in vivo and correlated TNF gene expression in vivo with the production of biologically active TNF by lymphoid cells in vitro. No significant difference in the expression of TNF mRNA was found between susceptible and resistant strains of mice during the course of infection. The depletion of CD4+ T cells in vitro did not change the level of TNF mRNA in BALB/c lymph node cells but led to the total disappearance of TNF mRNA in CBA mice. Unprimed spleen cells did not produce detectable amounts of TNF, whereas 1 week after infection, TNF bioactivity was detected and increased in both strains of mice until 5 weeks of infection. While neutralization of TNF activity in vivo did not alter the course of infection in BALB/c mice, in CBA mice it led to an increase in lesion size and a delay in the healing process but did not interfere significantly with the outcome of infection. Finally, no significant difference in the levels of lymphotoxin, TNF-RI, or TNF RII mRNA expression was found between both strains. The information resulting from these investigations supports the notion that, in vivo, TNF is not the decisive factor responsible for the resistant versus susceptible phenotype in leishmania infection.

Modulation of the transcripts for tumor necrosis factor-alpha and its receptors in vivo.

We investigated the effects of a single bacterial lipopolysaccharide (LPS) injection in vivo on the gene expression of tumor necrosis factor-alpha (TNF) and its receptors: TNF receptor type I (TNF-R 55 kDa or TNF-R1) and TNF receptor type II (TNF-R 75 kDa or TNF-R2) in various tissues and white blood cells. While TNF mRNA rapidly accumulated in most tissues, TNF-R1 and TNF-R2 mRNA levels were found to be differentially regulated in lung, spleen, lymph nodes and white blood cells. In most cases, TNF-R mRNA levels did not parallel TNF mRNA levels. These observations indicate that TNF-R of both types of capable of modulating the host response to LPS, not only by shedding of their extracellular domains, but also by strict regulation of their gene expression.

Profiles of cytokine production in relation with susceptibility to cerebral malaria.

Infection with Plasmodium berghei ANKA (PbA) leads, in susceptible strains of mice, to the development of cerebral malaria (CM), a lethal syndrome that reproduces some features of human CM. To study a possible relationship between genetic susceptibility to CM and the cytokine expression pattern, we quantitatively evaluated gene expression on RNA extracted from various organs of malaria-infected mice, using strains that are susceptible and resistant to CM. Northern blot analysis and semi-quantitative PCR showed that CM is associated with an increased TNF-alpha mRNA accumulation in the brain of mice developing the neurologic complications of CM. An increased IFN-gamma mRNA accumulation and a decreased expression of IL-4 and TGF-beta genes were also observed in mice susceptible to CM. In vitro restimulation studies using crude malarial Ag showed that lymphoid cell proliferation was higher in CM-susceptible than in CM-resistant infected mice. Moreover, susceptible mice produced large amounts of IFN-gamma, in a dose-dependent manner, in response to PbA Ag, whereas cells from resistant mice failed to produce significant amounts of this cytokine. Conversely, IL-2 and IL-4 production was significantly higher in infected CM-resistant mouse cells. No difference was seen in the production of IL-3 and IL-5 between resistant and susceptible PbA-infected mice. Upon stimulation with various malarial Ag, comparable amounts of TNF-alpha were produced by macrophages of either strain of mice. Taken together, these findings indicate that susceptibility to CM resides at the level of T cells rather than macrophages. Furthermore, the cytokine production profile is consistent with a predominant Th1-like response in mice developing cerebral complications of malaria.

Role of granulocyte-macrophage colony-stimulating factor in pulmonary fibrosis induced in mice by bleomycin.

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in pulmonary fibrosis elicited in mice by the intratracheal instillation of bleomycin was investigated by (1) evaluation of GM-CSF mRNA levels, (2) administration of GM-CSF, and (3) administration of anti-GM-CSF antibody. A significant increase of the GM-CSF mRNA level was evident in the lung RNA on day 5 after bleomycin instillation, but not on day 15. Abdominal infusion of GM-CSF (0.5 micrograms/h during days 7-15) did prevent the collagen deposition induced by bleomycin, as measured by the lung hydroxyproline content on day 15. In contrast, anti-GM-CSF antibody markedly aggravated the collagen deposition. On histological sections the proportion of lungs showing fibrosing alveolitis was decreased by GM-CSF and increased by anti-GM-CSF IgG. The percentage and number of macrophages within the bronchoalveolar lavage (BAL) fluid was increased by GM-CSF infusion and decreased by anti-GM-CSF antibodies. This study demonstrates that pulmonary GM-CSF has an inhibitory influence upon the alveolar remodeling and collagen deposition associated with pulmonary fibrosis.

Role of cytokines and adhesion molecules in malaria immunopathology.

Cerebral malaria (CM) is the most common cause of death in severe malaria; more than two million children die of CM annually. Although the mechanisms of this neurologic complication remain poorly understood, studies in an experimental model of CM suggest that a natural body protein seems to be a major cause of this deadliest complication of malaria, a finding that could point towards new methods of treatment. We have explored the pathogenesis of CM with particular attention to the possible relationship between susceptibility or resistance to CM and cytokine expression and secretion patterns. We found that CM is associated with an increased expression of tumor necrosis factor (TNF) and interferon (IFN)-gamma and a reduced expression of interleukin-4 (IL-4) and transforming growth factor (TGF)-beta. The data obtained are consistent with a predominantly Th1 response in mice developing the cerebral complications of malaria. The overexpression of TNF in brain was also correlated with the augmented expression of adhesion molecules involved in the sequestration of leukocytes in brain vessels, a distinctive feature of CM. These observations were seen in relation to the immune status of man, in which, akin to the mouse model, a predominant Th1 response and upregulation of adhesion molecules in brain endothelium appear to be associated with susceptibility to the neurological complications of CM.

Tumor necrosis factor alpha is involved in mouse growth and lymphoid tissue development.

Tumor necrosis factor alpha (TNF-alpha), a major mediator of inflammation, also possesses a wide pleiotropism of actions, suggesting its involvement in physiological conditions. TNF-alpha mRNA is present in mouse embryonic tissues and also in fetal thymus and spleen. Repeated injections of a monospecific polyclonal rabbit anti-mouse TNF-alpha antibody in mice, starting either during pregnancy or at birth, led to a severe but transient growth retardation, already present at birth, reaching a 35% decrease in body weight at 3 wk, with complete recovery at 8 wk. The insulin growth factor I (IGF-I) blood levels were decreased to about 50%; growth hormone release and other endocrine functions were unaltered. A marked atrophy of the thymus, spleen, and lymph nodes was also observed, with lymphopenia and impaired development of T and B cell peripheral lymphoid structures. The pathways involving TNF-alpha in IGF-I release and early body growth are probably distinct from those by which TNF-alpha participates in early development of lymphoid tissues, where its low physiological release may contribute to enhance lymphoid cell expansion.

Effect of cyclosporin A and zidovudine on immune abnormalities observed in the murine acquired immunodeficiency syndrome.

Two therapeutic modalities, zidovudine (targeting retroviral replication) and cyclosporin A (targeting immunopathologic consequences of retroviral expression) were evaluated in a murine model of AIDS. In previous studies, cyclosporin A treatment (40 or 60 mg/kg/day) before and after infection with LP-BM5 murine leukemia viruses protected against the development of immunodeficiency disease. The present study extends these findings. First, a low dose of cyclosporin A (20 mg/kg/day) was ineffective, and treatment initiated 5 days after infection did not protect against virus-induced lymphoproliferation and hypergammaglobulinemia. Second, zidovudine added to drinking water (0.1 mg initiated 5 days after infection and continued for 8 weeks) was more effective than 0.2 mg/mL given day 5-12 after infection. This treatment reduced lymph node size, disease severity as determined histologically, retrovirus-induced gp70 expression, and IgE (but not IgM and IgG) levels. Third, combined treatment had an additive, protective effect on lymphocyte proliferative capacity. This successful dual therapeutic strategy in a mouse model has potential applicability for similar approaches in treating human immunodeficiency virus infection.

Significance of cytokine production and adhesion molecules in malarial immunopathology.

The pathological expression in malaria infection depends largely on immunopathologic responses induced by the parasite. In the past few years, we have attempted to analyze mechanisms by which inappropriate immune response to some malarial antigens can generate major complications of malaria and particularly neurovascular lesions. To this end, we have undertaken a study aimed at a more precise definition of immunopathological parameters of malaria infection, and more particularly those involved in cerebral malaria (CM). CM, the most severe complication of falciparum infection in man, represents a major problem of public health at the world level.