Cardiocerebral protective effects of dexmedetomidine as anesthetic in colorectal cancer surgery.

OBJECTIVE: To explore the cardiocerebral protective effect of dexmedetomidine as an anesthetic in colorectal cancer surgery. PATIENTS AND METHODS: A total of 246 colorectal cancer patients were enrolled in this retrospective analysis. Those patients were admitted to the Affiliated Hospital of Qingdao University and underwent surgery from July 2014 to July 2016. The patients were divided into observation group and control group according to the anesthetic used in surgery. The conventional anesthetic was administered to patients in control group, whereas conventional anesthetic supplemented with dexmedetomidine was administered to patients in the observation group. The heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), jugular venous oxygen saturation (Sj-vO2), cerebral oxygen extraction ratio (ERO2), and cerebral arterial partial pressure of oxygen (PaO2) were recorded before dexmedetomidine administration (T0), 30 min after start of surgery (T1), and 2 h after surgery (T2). Central venous blood (4 ml) was withdrawn 6 hours and 24 hours after surgery. Following centrifugation, the serum was collected and stored at -70°C. After collection of all the blood samples, concentrations of creatine kinase (CK-MB), troponin I (cTnI), TNF-α and S100ß in serum were measured using ELISA, and differences between the two groups were compared. RESULTS: Differences of the parameters measured at T0 were not statistically significant between observation group and control group (p>0.05), whereas the parameters measured at T1 and T2 were significantly better in the observation group than those in the control group (p<0.05). The post-surgery blood test showed that indicators of cardiocerebral hemodynamics were better in the observation group than those in the control group (p<0.05). CONCLUSIONS: Administration of dexmedetomidine in colorectal cancer surgery can provide effective cardiocerebral protection and it is worth popularizing in clinical practice.

LECT2 association with macrophage-mediated killing of Helicobacter pylori by activating NF-κB and nitric oxide production.

Helicobacter pylori employs unique methods to colonize the stomach, which induces chronic inflammation. It is also able to avoid eradication by macrophages and other immune cells. Leukocyte cell-derived chemotaxin 2 (LECT2), a multi-functional cytokine involved in many pathological conditions, has recently been shown to activate macrophages via the CD209a receptor. Therefore, we aimed to investigate the effects of LECT2 on H. pylori-infected macrophages. Macrophages were treated with recombinant LECT2, and both their ability to kill H. pylori and produce nitric oxide were analyzed. Western blot was performed to determine nuclear translocation and protein phosphorylation of p65, a subunit of nuclear factor (NF)-κB. Transfection experiments were performed to analyze the signaling pathway of LECT2 in macrophages. We found that treatment with LECT2 enhanced H. pylori killing and nitric oxide production in macrophages. In addition, DNA-binding activity and nuclear translocation of p65 were up-regulated by LECT2 treatment. Furthermore, we found that NF-κB activation by LECT2 was mediated by Raf-1 in macrophages, and Raf-1 phosphorylation was specifically altered in response to LECT2. Moreover, LECT2 induced Ser28 phosphorylation in the intracellular domain of CD209a. CD209a Ser28 phosphorylation was required for LECT2-induced Raf-1 and NF-κB activation in RAW264.7 macrophages. Our study showed that the effects of LECT2 on H. pylori killing and nitric oxide production were dependent on CD209a phosphorylation, Raf-1, and NF-κB activation. Together, these results demonstrate for the first time that exposure to LECT2 can modulate specific intracellular mechanisms downstream of CD209a to enhance H. pylori killing and nitric oxide production in macrophages.

IFNgamma deficient C57BL/6 (H-2b) mice develop collagen induced arthritis with predominant usage of T cell receptor Vbeta6 and Vbeta8 in arthritic joints.

BACKGROUND: Transgenic deficiency in interferon gamma (IFNgamma) or IFNgamma receptor makes resistant strains of mice bearing H-2(b) or H-2(d) susceptible to collagen induced arthritis (CIA). OBJECTIVE: To determine whether the escape from regulation of disease susceptibility at the major histocompatibility complex level involves a new use of autoimmune T cells expressing T cell receptor (TCR) Vbeta that vary from the cell populations previously identified within arthritic joints. METHODS: Arthritis was induced by a standard protocol with type II bovine collagen (CII) in complete Freund's adjuvant. Clinical features, histopathology, immunological responses, and TCR profile in arthritic joints in IFNgamma knockout C57BL/6 (B6.IFNgamma KO) mice (H-2(b)) were compared directly with those in DBA/1 mice (H-2(q)). RESULTS: 60-80% of B6.IFNgamma KO mice developed a progressive arthritis with a similar clinical course to classical CIA in DBA/1 mice. The affected joints in B6.IFNgamma KO mice had an erosive form of arthritis with similar features to joint disease in DBA/1 mice. B6.IFNgamma KO mice produced significantly higher levels of IgG2b and IgG1 autoantibodies to murine CII and showed increased proliferative response to CII compared with B6 mice. Comparable levels of interleukin 1beta and tumour necrosis factor alpha expression were detected in arthritic joints from beta6.IFNgamma KO and DBA/1 mice. B6.IFNgammaKO mice used predominantly TCR Vbeta6 and Vbeta8 in arthritic joints. This TCR Vbeta profile is similar to that found in DBA/1 mice with CIA. CONCLUSIONS: C57BL/6 mice deficient in IFNgamma production can develop arthritis that resembles classical CIA. These data suggest that IFNgamma is a key factor mediating susceptibility to CIA.

Interferon gamma eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells.

In Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected wild-type mice, there was a large expansion of an activated (CD44(hi)) splenic CD4 T cell population followed by a rapid contraction of this population to normal numbers. Contraction of the activated CD4 T cell population in wild-type mice was associated with increased apoptosis of activated CD4 T cells. In BCG-infected interferon (IFN)-gamma knockout (KO) mice, the activated CD4 T cell population did not undergo apoptosis. These mice accumulated large numbers of CD4(+)CD44(hi) T cells that were responsive to mycobacterial antigens. Addition of IFN-gamma to cultured splenocytes from BCG-infected IFN-gamma KO mice induced apoptosis of activated CD4 T cells. IFN-gamma-mediated apoptosis was abolished by depleting adherent cells or Mac-1(+) spleen cells or by inhibiting nitric oxide synthase. Thus, IFN-gamma is essential to a regulatory mechanism that eliminates activated CD4 T cells and maintains CD4 T cell homeostasis during an immune response.

Failure to suppress the expansion of the activated CD4 T cell population in interferon gamma-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis.

Mice deficient in interferon (IFN)-gamma or IFN-gamma receptor develop progressive and fatal experimental autoimmune encephalomyelitis (EAE). We demonstrate that CD4 T cells lacking IFN-gamma production were required to passively transfer EAE, indicating that they were disease-mediating cells in IFN-gamma knockout (KO) mice. IFN-gamma KO mice accumulated 10-16-fold more activated CD4 T cells (CD4(+)CD44(hi)) than wild-type mice in the central nervous system during EAE. CD4(+)CD44(hi) T cells in the spleen and central nervous system of IFN-gamma KO mice during EAE showed markedly increased in vivo proliferation and significantly decreased ex vivo apoptosis compared with those of wild-type mice. IFN-gamma KO CD4(+)CD44(hi) T cells proliferated extensively to antigen restimulation in vitro and accumulated larger numbers of live CD4(+) CD44(hi) T cells. IFN-gamma completely suppressed proliferation and significantly induced apoptosis of CD4(+)CD44(hi) T cells responding to antigen and hence inhibited accumulation of live, activated CD4 T cells. We thus present novel in vivo and in vitro evidence that IFN-gamma may limit the extent of EAE by suppressing expansion of activated CD4 T cells.

Differential activities of immunogenic collagen type II peptides in the induction of nasal tolerance to collagen-induced arthritis.

Nasal tolerance has recently been used to modulate immune responses in animal models of autoimmunity. We have compared immunogenic collagen type II (CII) peptides for induction of nasal tolerance in DBA/1 mice to collagen-induced arthritis (CIA). Three synthetic peptides corresponding to T cell-stimulating sequences of alpha1(II)-CB11, 260-270, 245-270 and 259-273, one peptide analog 245-270 (A260B261N263) and one myelin basic protein (MBP) peptide 89-101 were administered intranasally to DBA/1 mice respectively (total 300 microg peptide/mouse on three consecutive days) 10 days prior to CII immunization. Forty percent of CII245-270 (P<0.05) and 20% CII260-270 (P>0.05) treated mice did not develop arthritis whilst all of the mice treated with CII245-270 (A260B261N263) or CII259-273 developed arthritis compared to those in control groups (PBS- and MBP89-101-treated). The mice in either the CII245-270- or CII260-270-treated group which developed arthritis had a significantly delayed onset and their disease was less severe both clinically and histologically. All mice in both CII245-270- and CII260-270-treated groups had a reduced serum level of anti-CII antibody (P<0.01), with a marked reduction of IgG2a. Drain lymph node (LN) cells taken 7 days after CII immunization from these mice showed a significant reduction of interferon (IFN)-gammaP<0.01) production uponin vitro stimulation with CII. These results indicate that intranasal administration of synthetic CII peptides can control CIA, which is achieved by down-regulating the Th1 CII-induced responses. In addition, they stress that a fine 'tuning' of the peptide able to induce 'tolerance' is required to achieve the optimal effect.

Treatment of a newly established transgenic model of chronic arthritis with nondepleting anti-CD4 monoclonal antibody.

We established a novel animal model for rheumatoid arthritis (RA) by following backcrossing to DBA/1 of (SWR/J x DBA/1)F1 TCR-beta Tg mice, previously reported to be highly susceptible to collagen-induced arthritis. These mice evolved, upon collagen type II immunization, into a chronic arthritis that histopathologically resembles RA. The availability of such a model prompted us to study the role of CD4+ T cells throughout the evolution of disease. Here, we show that administration of nondepleting anti-CD4 not only prevented the evolution of disease but also treated established arthritis. Moreover, functional analyses of T cells isolated from anti-CD4-treated mice demonstrated that the mechanism of protection is not achieved by suppression of the Th1 population but is mediated by induction of collagen type II-specific T cell anergy. Our study suggests that: 1) CD4+ T cells have a fundamental role both in the induction and in the perpetuation of disease; 2) targeting T cells may be an appropriate therapeutic option; and 3) a suitable and well-balanced anti-CD4 treatment may be a valid approach to the control of RA.

Induction of Th2 cytokines and control of collagen-induced arthritis by nondepleting anti-CD4 Abs.

CD4+ T cells play a key role in the development of cell-mediated autoimmune diseases, and their modulation has been used to prevent autoimmune diseases in animal models. The effect of the nondepleting anti-CD4 mAb (KT6) was investigated in collagen-induced arthritis (CIA) in DBA/1 mice and in adoptive transfer of CIA into SCID mice. KT6 (200 microg/dose/mouse) was administered systematically from the day of collagen type II (CII) immunization and continued by alternate-day injection until day 11. Only 20% of KT6-treated mice developed arthritis compared with 100% of the isotype control treated mice (p < 0.001). KT6 treatment in a similar regimen also abrogated the adoptive transfer of CIA into SCID mice. Serum levels of IgG2a anti-CII Abs in KT6-treated DBA/1 mice were significantly reduced (p < 0.001), while IgG1 anti-CII were not significantly changed (p > 0.05). Lymph node cells of mice treated in vivo with KT6 had a reduced production of IFN-gamma but increased IL-4 upon in vitro challenge with CII. Furthermore, KT6 could also reverse the profile of cytokine release of in vivo primed and pathogenic CII-specific T cells. These results demonstrate that in vivo modulation with nondepleting anti-CD4 Abs prevents CIA, likely by altering the functional profile of Th1 T cells to Th2. Furthermore, we demonstrate that this treatment can not only prevent, but more importantly also control pathogenic T cells by switching their cytokine production from a Th1 to a Th2-like profile. Our results thus provide compelling evidence of how treatment with nondepleting anti-CD4 may control an autoimmune process. They also indicate that this approach not only prevents, but also could control ongoing autoimmune diseases.

Expression of cyclooxygenase-2 in human and an animal model of rheumatoid arthritis.

An inducible form of cyclooxygenase-2 (COX-2) has been shown to be upregulated in vitro by various pro-inflammatory agents, such as lipopolysaccharide, IL-1 and TNF, COX-2 appears to be responsible for the increase in prostaglandin synthesis at the site of inflammation. To examine the involvement of COX-2 in inflammation, we analysed the expression of this gene in human rheumatoid arthritis (RA) and in rat adjuvant-induced arthritis. Immunocytochemical studies of synovial membrane biopsies from human RA, osteoarthritic (OA) and normal joints using a COX-2 specific antibody showed positive staining in RA, but not in normal synovial membranes. Specifically, expression of COX-2 was detected in synovial lining cells, lymphoid aggregates and endothelial cells of blood vessels. Although some positive staining was observed in the OA joints, the number of stained cells was dramatically lower and the staining of the cells was less intense than in the rheumatoid tissue. By reverse transcription and polymerase chain reaction analysis, COX-2 mRNA was detected in the rat adjuvant arthritic limb, whereas no COX-2 mRNA was detectable in the normal limb. These observations indicate that COX-2 expression is upregulated in inflammatory joint disease and that COX-2 is a potential therapeutic target for specific inhibition.

Transfer of type II collagen-induced arthritis from DBA/1 to severe combined immunodeficiency mice can be prevented by blockade of Mac-1.

Collagen-induced arthritis in susceptible mice is widely accepted as an experimental model for human rheumatoid arthritis (RA). We have investigated the role of the Mac-1 integrin beta 2 in the development and maintenance of arthritis by means of in vivo administration of 5C6 monoclonal antibody (mAb) to block this receptor. Injection of a single dose of 5C6 mAb (0.5 mg, intraperitoneally) prior to the expected onset of collagen-induced arthritis in DBA/1 mice diminished the severity of subsequent disease in these animals, as assessed both clinically and histologically (P < 0.01, chi 2). In the DBA/1 to severe combined immunodeficiency (SCID) transfer model of arthritis, the incidence of clinical arthritis was significantly reduced in SCID mice receiving maintained 5C6 treatment commencing the day prior to administration of donor splenocytes. Histological evaluation of joints from animals without clinically evident arthritis confirmed the absence of an inflammatory infiltrate in 22/27 joints examined. In a minority of these joints, however, synovial hyperplasia was apparent. In contrast, delaying antibody administration until 10 days after donor spleen cell transfer failed to protect three of five SCID recipients. These results confirm a functional role for Mac-1 in the generation of collagen-induced arthritis in mice. Since mAb 5C6 is non-cytotoxic, its action must be by blockade of the interactions between Mac-1 and its natural ligand(s). Our findings support the hypothesis that cells expressing Mac-1 play an important role in the induction and maintenance of joint damage in collagen-induced arthritis.

High level of interleukin-10 production by the activated T cell population within the rheumatoid synovial membrane.

OBJECTIVE: To characterize the cytokine profile of the activated T cell population derived from the synovial membrane of rheumatoid arthritis (RA) patients. METHODS: Interleukin-2 (IL-2) was used to select for in vivo-activated T cells from the synovial membrane of 2 patients with RA, and the cells were cloned nonspecifically. The cytokine production profile of these clones was compared with that of clones derived from peripheral blood monocytes (PBM) by stimulating all clones for 24 hours with immobilized anti-CD3 (coated at 10 micrograms/ml) or phorbol-12-myristate-13-acetate (10 ng/ml) plus soluble anti-CD3 (1 microgram/ml). Interferon-gamma (IFN gamma), IL-4, and IL-10, the cytokines that discriminate between Th1 and Th2 cells and are involved in immunoregulation, were assayed by enzyme-linked immunosorbent assay. RESULTS: There was a difference in the cytokines produced by the clones derived from the rheumatoid membranes compared with clones derived from the periphery. Clones derived from both membranes and PBM were mostly IFN gamma-producers, i.e., either a Th0 or a Th1 profile. There was a high proportion of IFN gamma/high IL-10-producing cells derived from the joint, but not from the periphery. Of clones derived from the synovial membrane of each of 2 RA patients, 100% and 50% produced both 1-10 ng/ml IFN gamma and > 7 ng/ml IL-10, compared with < 7% of clones derived from normal or RA peripheral blood. In addition, when autologous membrane and PBM were compared, the mean concentration of IL-10 produced by the clones derived from the synovial membrane sample was significantly different from those produced by clones derived from peripheral blood (P < 0.02). CONCLUSION: The cytokine profile of the T cell clones that were obtained from the RA joint after expansion with IL-2 is distinct from that of the T cells that are predominant in PBM. This supports the concept that the T cells subsets that accumulate in the joint are not a random sample. The high level of IL-10 production by clones derived from the synovium suggests that this cytokine may be a major contributor to the endogenous immunosuppression that occurs in RA.

Monoclonal anti-TNF alpha antibody as a probe of pathogenesis and therapy of rheumatoid disease.

Rheumatoid arthritis is a common cause of chronic disability for which current therapies are of limited value in controlling the disease process and outcome. Our initial approach to understanding the pathogenesis of RA and defining a novel therapeutic target was to investigate the role of cytokines by blocking their action with antibodies on cultured synovial-derived mononuclear cells in vitro. These investigations suggested that neutralization of TNF alpha with antibodies significantly inhibited the generation of other pro-inflammatory cytokines also over-produced, such as, IL-1, GM-CSF, IL-6 and IL-8. The implication that blockade of a single cytokine, TNF alpha might have far-reaching effects on multiple cytokines and thereby exert significant anti-inflammatory and protective effects on cartilage and bone of joints, was tested in arthritic DBA/1 mice immunized with collagen II. Impressive amelioration of joint swelling and joint erosions in this model encouraged clinical trials with a monoclonal anti-TNF alpha antibody. The cA2 chimeric anti-TNF alpha high-affinity antibody was initially tested in an open-label study at a dose of 20 mg/kg on 20 patients, with substantial and universal benefit. Subsequently, a randomized placebo-controlled double-blind trial was performed on 73 patients comparing a single intravenous injection of placebo (0.1% human serum albumin) with two doses of cA2. Using a composite disease activity index, at 4 weeks post infusion, 8% of patients receiving placebo improved compared with 44% receiving 1 mg/kg cA/2 and 79% receiving 10 mg/kg. Between 2 to 4 repeated cycles of cA2 were administered to 7 patients and all patients showed improvement of a similar magnitude with each cycle. These data support our proposition that TNF alpha is implicated in the pathogenesis of RA, and is thus a key therapeutic target. Monoclonal anti-TNF alpha antibodies control disease flares and are candidate agents for longer-term control of RA, although repeated therapy with cA2 is associated with anti-idiotypic responses in 50% of patients and a trend toward shortening of the duration of response. In the DBA/1 arthritic mice, synergy of action of anti-TNF and anti-CD4 is observed together with suppression of an anti-globulin response, indicating one way in which benefit might be augmented in the future.

A rare mediastinal tumour presenting with systemic effects due to IL-6 and tumour necrosis factor (TNF) production.

Patients presenting with prolonged systemic illnesses with no specific clinical or serological defining features may be diagnosed as having atypical systemic vasculitides, but often turn out to have occult malignancies. Cytokines have been implicated in causing many of the systemic effects in such cases. In this study we describe a patient presenting after 2 years of a severe systemic illness with a marked acute phase response, due to an occult mediastinal angiomatoid malignant fibrous histiocytoma. Tumour resection was curative. We evaluated in detail the local and systemic production of cytokines induced by this tumour. Blood samples were taken pre- and post-operatively for cytokine studies. In vitro production of IL-2, IL-2R, IL-1 beta, IL-6 and TNF-alpha by cultured monocytes from the patient, as well as plasma cytokine levels, were measured by ELISA. Tumour cytokine production was also evaluated immunocytochemically, and by in situ hybridization with specific cDNA probes. Plasma IL-2R and IL-6, and IL-6 and TNF-alpha production by peripheral blood monocytes were markedly elevated before tumour resection, normalizing post-operatively. Most tumour cells and infiltrating lymphocytes stained with antibodies to IL-6, IL-6R and TNF-alpha, and expressed HLA class II. IL-6 and TNF-alpha mRNA production in the tumour was confirmed by in situ hybridization studies. We have described the first case of an occult angiomatoid malignant fibrous histiocytoma in the mediastinum. Studies of cytokine expression suggested that chronic TNF, IL-6, and IL-2 production by leucocytes and tumour cells in this patient was responsible for the severe systemic illness with which she presented.

Immunoregulatory role of interleukin 10 in rheumatoid arthritis.

The presence and the role of interleukin 10 (IL-10), a potent cytokine synthesis inhibitory factor and antiinflammatory cytokine, were investigated in rheumatoid arthritis (RA). The expression of both mRNA and protein for IL-10 could be demonstrated in RA and osteoarthritis (OA) joints. Human IL-10 mRNA could be demonstrated by polymerase chain reaction amplification of cDNA made by reverse transcription of total RNA extracted directly from synovial tissue in five out of five RA and four out of five OA patients. IL-10 protein was demonstrated by specific immunoassay and immunohistology. IL-10 protein was spontaneously produced in all 11 RA and 17 OA synovial membrane cultures investigated, and this production was sustained for up to 5 d in culture in the absence of any extrinsic stimulation. IL-10 protein could also be detected by immunohistology in all five RA and four OA synovial membrane biopsies investigated, but not three normal synovial membranes. Immunohistology revealed that the IL-10 was localized to the synovial membrane lining layer and mononuclear cell aggregates. Immunofluorescence double staining revealed that the sources of IL-10 were monocytes in the lining layer, and T cells in the mononuclear cell aggregates. We found evidence that the IL-10 expression was functionally relevant, as neutralization of endogenously produced IL-10 in the RA synovial membrane cultures resulted in a two- to threefold increase in the protein levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and IL-1 beta, although IL-6 and IL-8 levels were not affected. The addition of exogenous recombinant IL-10 to the RA synovial membrane cultures resulted in a two- to threefold decrease in the levels of TNF-alpha and IL-1 beta. IL-8 levels were reduced by day 5; however, IL-6 levels were not affected by exogenous IL-10. Neutralization of the endogenous IL-10 in two out of seven RA synovial membrane cultures resulted in the expression of detectable levels of interferon gamma (561-1,050 pg/ml). Taken together, the above findings suggest that IL-10 is spontaneously produced in RA and OA and is an important immunoregulatory component in the cytokine network of RA, regulating monocyte and in some cases T cell cytokine production.

Localisation of interleukin 8 in the synovial membrane, cartilage-pannus junction and chondrocytes in rheumatoid arthritis.

Interleukin-8 (IL-8) may play an important role in the development of synovitis in rheumatoid arthritis (RA), in that it is a powerful chemoattractant for neutrophils and T cells. The aim of this study was to examine the distribution of IL-8 in the synovial membrane and cartilage, from RA, osteoarthritis (OA) and normal joints. By immunohistochemical techniques, IL-8 was shown to be present in the lining layer cells in RA (87%) and in OA (62%). By contrast, only a few of the normal synovial lining layer cells (14%) contained IL-8. Deeper in the membrane the number of IL-8 positive cells decreased. Only vessels were highly positive for IL-8. At the RA cartilage-pannus junction 26% of the cells contained IL-8, whereas at the OA cartilage-pannus junction 8% of the cells were IL-8 positive (P < 0.05). Chondrocytes present in joint surface cartilage stained positive for IL-8 in an average of 20% of the cells of both RA and OA. These results provide histological evidence that IL-8 is present in the arthritic synovial tissue and cartilage, and is distributed in a manner that may form a chemotactic gradient, which favours localisation of neutrophils to the joint lumen.

Localization of tumour necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kD TNF receptor.

The distribution of TNF-alpha, p55 TNF receptor (TNF-R) and p75 TNF-R in normal skin and uninvolved and lesional skin from psoriasis patients has been investigated, using specific mono- and polyclonal antibodies. In normal skin, and uninvolved and lesional skin from psoriasis patients, p55 TNF-R is associated with epidermal keratinocytes and a network of upper dermal dendritic cells. This suggests that the actions of TNF-alpha on epidermal cells in vivo are mediated by binding to the p55 TNF-R. In lesional psoriasis skin, there was staining of the parakeratotic stratum corneum and increased expression of p55 TNF-R in association with upper dermal blood vessels. Staining for p75 TNF-R in normal skin was restricted to eccrine sweat ducts and dermal dendritic cells, and was absent from the epidermis. In lesional psoriasis skin, there was staining for p75 TNF-R in association with upper dermal blood vessels and perivascular infiltrating cells. TNF-alpha in normal skin was predominantly localized to the basal cell layers of the epidermis, and was seen in association with eccrine ducts and sebaceous glands. In lesional psoriasis skin, and to a lesser extent in uninvolved psoriasis skin, TNF-alpha was distributed throughout the epidermis, and was also specifically localized to upper dermal blood vessels. Up-regulation of TNF-alpha, p55 TNF-R and p75 TNF-R on dermal blood vessels in psoriasis may play an important role in the pathogenesis of this condition by promoting cutaneous recruitment of inflammatory cells.

TNF-alpha in rheumatoid arthritis and prospects of anti-TNF therapy.

Our work has shown that TNF alpha is produced by cultured mononuclear cells from rheumatoid arthritis joints and appears to regulate the production of IL-1. Immunohistochemical examination has shown the presence of TNF alpha in the synovium, e.g. in the lining layer, some endothelial cells and most importantly, in the cells in the cartilage pannus junction. TNF receptors (both p55 and p75) have a similar distribution, thereby suggesting that TNF has the potential for autocrine and paracrine activity in the joint. The concept that TNF alpha is pathogenic in inflammatory arthritis has been validated by showing that neutralizing monoclonal anti-TNF antibodies significantly attenuate collagen-induced arthritis in mice. In preliminary trials in rheumatoid patients anti-TNF appears to have an impressive effect on indices of disease activity including C-reactive production and serum amyloid-A production. TNF alpha appears to be a relevant therapeutic target in rheumatoid disease.

Detection of cytokines at the site of tuberculin-induced delayed-type hypersensitivity in man.

Cytokines are chiefly local mediators which play an important role in the regulation of the cell-cell interactions which may be involved in the development of the delayed-type hypersensitivity (DTH) reaction. Using immunohistochemical techniques, the presence of IL-1 alpha, IL-1 beta, IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) in the skin in tuberculin-purified protein derivative (PPD)-induced DTH reactions was investigated in six normal individuals. Cells staining for these cytokines were first observed 6 h after PPD challenge, and they were detected throughout the duration of the 7-day experiment. The number of cells staining for IFN-gamma reached a peak at 48 h, where 33% of the total aggregate cells were positive, but declined thereafter to 3% at day 7. On the other hand, the number of cells staining for TNF-alpha and IL-1 persisted at high levels throughout the observation period of 7 days (e.g. at 48 h and thereafter, about 40% cells positive for TNF-alpha and 20% for IL-1 alpha and IL-1 beta). Double immunofluorescence and staining on sequential sections showed that IFN-gamma-staining cells were CD3+ T cells; TNF-alpha, IL-1 and IL-6 staining cells were mainly of the CD68+ macrophages/monocytes and that 80% of the CD1a+ cells (Langerhans-like cells) in the dermis contained TNF-alpha and IL-1. The presence of these cytokines at the site of inflammation suggests that they may be locally produced by the inflammatory cells. Their persistence during the reaction suggests that they are intimately associated with this response, and are involved in the development of the reaction.

Localization of interleukin-1 alpha, type 1 interleukin-1 receptor and interleukin-1 receptor antagonist in the synovial membrane and cartilage/pannus junction in rheumatoid arthritis.

Using monoclonal antibodies and immunohistochemical techniques we have investigated the presence and distribution of interleukin-1 alpha (IL-1 alpha), type 1 IL-1 receptor (IL-1R1) and of interleukin-1 receptor antagonist (IL-1ra) in synovial tissue from 18 rheumatoid arthritis (RA) and eight osteoarthritis (OA) patients and in eight normal synovial tissue samples. IL-1 alpha and IL-1R1 were found in all of the samples examined. In RA, there were a large number of synovial cells expressing IL-1 alpha and IL-1R1, with 85 and 90% positive cells in the lining layer, 45 and 80% in the interaggregate area, and 90% of the vascular endothelial cells. In the lymphoid aggregates, 20% of the cells contained IL-1 alpha and 70% expressed IL-1R1. IL-1 alpha and IL-1R1 expressing cells showed a similar distribution in OA synovial membrane, but there was a smaller number of positive cells in the deeper area; and the staining intensity was lower. In contrast to IL-1 alpha and IL-1R1, IL-1ra was found only in 10/18 RA, 5/8 OA and 2/8 normal tissue samples. IL-1ra was detected in 35% of RA and 45% OA lining layer cells; 25% RA and 35% OA vascular endothelium; 10% RA and 15% OA interstitial cells and 30% cells in RA lymphoid aggregate. The staining intensity in both RA and OA tissues was comparably low. The presence of IL-1ra in RA and OA tissues was confirmed by Northern blot analysis for IL-1ra mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of cytokines at the cartilage/pannus junction in patients with rheumatoid arthritis: implications for the role of cytokines in cartilage destruction and repair.

Cytokine release at the cartilage/pannus junction (CPJ) may be involved in cartilage destruction and tissue repair in rheumatoid arthritis (RA). Tissue samples of CPJ from 12 RA patients were examined for the presence of cytokines using immunohistochemical techniques with immunoaffinity purified F(ab')2 antibodies raised against recombinant human cytokines. Twenty-four areas of distinct CPJ at which a discrete junction between cartilage and overlying pannus exists were observed. In all specimens, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha. IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta 1 were detected in cells in pannus particularly along the surface of cartilage and at the site of cartilage erosion. Double immunofluorescence staining showed that most cytokine containing cells also labelled with a macrophage marker (CD68). About 50% of blood vessel endothelial cells stained for GM-CSF. Twelve areas of diffuse fibroblastic CPJ, at which an indistinct margin is seen between cartilage and pannus were examined. At this site, TGF-beta 1 was the only cytokine detected in fibroblast-like cells. None of these cytokines were detected in synovial tissue at the normal synovium/cartilage junction. Chondrocytes from all 11 normal specimens as well as those from RA patients stained for IL-1 alpha, TNF-alpha, IL-6, GM-CSF and TGF-beta 1, especially those close to subchondral bone. However, IL-1 beta, interferon-gamma and lymphotoxin were not detected in either the normal synovium/cartilage junction or rheumatoid CPJ.(ABSTRACT TRUNCATED AT 250 WORDS)