Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis

Despite its potent ability to inhibit proinflammatory cytokine synthesis, interleukin (IL)-10 has a marginal clinical effect in rheumatoid arthritis (RA) patients. Recent evidence suggests that IL-10 induces monocyte/macrophage maturation in cooperation with macrophage-colony stimulating factor (M-CSF). In the present study, we found that the inducible subunit of the IL-10 receptor (IL-10R), type 1 IL-10R (IL-10R1), was expressed at higher levels on monocytes in RA than in healthy controls, in association with disease activity, while their expression of both type 1 and 2 tumour necrosis factor receptors (TNFR1/2) was not increased. The expression of IL-10R1 but not IL-10R2 was augmented on monocytes cultured in the presence of RA synovial tissue (ST) cell culture supernatants. Cell surface expression of TNFR1/2 expression on monocytes was induced by IL-10, and more efficiently in combination with M-CSF. Two-color immunofluorescence labeling of RA ST samples showed an intensive coexpression of IL-10R1, TNFR1/2, and M-CSF receptor in CD68⁺ lining macrophages. Adhered monocytes, after 3-day preincubation with IL-10 and M-CSF, could produce more IL-1β and IL-6 in response to TNF-α in the presence of dibutyryl cAMP, as compared with the cells preincubated with or without IL-10 or M-CSF alone. Microarray analysis of gene expression revealed that IL-10 activated various genes essential for macrophage functions, including other members of the TNFR superfamily, receptors for chemokines and growth factors, Toll-like receptors, and TNFR-associated signaling molecules. These results suggest that IL-10 may contribute to the inflammatory process by facilitating monocyte differentiation into TNF-α-responsive macrophages in the presence of M-CSF in RA.     

Immunostimulating activity by polysaccharides isolated from fruiting body of <Emphasis Type="Italic">Inonotus obliquus</Emphasis>

In this study, we investigated the immunostimulating activity of polysaccharides isolated from fruiting body of Inonotus obliquus (PFIO). Additionally, the signaling pathway of PFIO-mediated macrophage activation was investigated in RAW264.7 macrophage cells. We found that PFIO was capable of promoting NO/ROS production, TNF-α secretion and phagocytic uptake in macrophages, as well as cell proliferation, comitogenic effect and IFN-γ/IL-4 secretion in mouse splenocytes. PFIO was able to induce the phosphorylation of three MAPKs as well as the nuclear translocation of NF-κB, resulting in activation of RAW264.7 macrophages. PFIO also induced the inhibition of TNF-α secretion by anti-TLR2 mAb, consequently, PFIO might be involved in TNF-α secretion via the TLR2 receptor. In addition, our results showed that oral administration of PFIO suppressed in vivo growth of melanoma tumor in tumorbearing mice. In conclusion, our experiments presented that PFIO effectively promotes macrophage activation through the MAPK and NF-κB signaling pathways, suggesting that PFIO may potentially regulate the immune response.     

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80⁺ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80⁺ macrophages and CD11c⁺ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4⁺ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.     

Repeated Stress Down-Regulates β<Subscript>2</Subscript>- and α<Subscript>2C</Subscript>-Adrenergic Receptors and Up-Regulates Gene Expression of IL-6 in the Rat Spleen

Catecholamines are among first compounds released during stress, and they regulate many functions of the organism, including immune system, via adrenergic receptors (ARs). Spleen, as an immune organ with high number of macrophages, possesses various ARs, from which β₂-ARs are considered to be the most important for the modulation of immune functions. Nevertheless, little is known about the regulation and involvement of ARs in the splenic function by stress. Therefore, the aim of this work was to measure the gene expression of ARs and several cytokines in the spleen of rats exposed to a single and repeated (14×) immobilization stress (IMO). We have found a significant increase in β₂-AR mRNA after a single IMO, but a significant decrease in β₂-AR mRNA and protein level after repeated (14×) IMO. The most prominent decrease was detected in the gene expression of the α_2A- and α_2C-AR after repeated IMO. However, changes in mRNA were translated into protein levels only for the α_2C-subtype. Other types of ARs remained unchanged during the stress situation. Since we proposed that these ARs might affect production of cytokines, we measured gene expression of pro-inflammatory (TNF-α, IL-1β, IL-6 and IL-18) and anti-inflammatory (IL-10 and TGF-β1) cytokines. We detected changes only in IL-6 and IL-10 mRNA levels. While IL-6 mRNA was increased, IL-10 mRNA dropped after repeated IMO. According to these results we suggest that changes of β₂- and α_2C-ARs participate in IL-6-mediated processes in the spleen, especially during chronic stress situations.     

<Emphasis Type="Italic">S</Emphasis>-Propargyl-cysteine (SPRC) attenuated lipopolysaccharide-induced inflammatory response in H9c2 cells involved in a hydrogen sulfide-dependent mechanism

The present study attempts to investigate the effects of S-propargyl-cysteine (SPRC), a sulfur-containing amino acid, on lipopolysaccharide (LPS)-induced inflammatory response in H9c2 cardiac myocytes. We found that SPRC prevented nuclear factor-κB (NF-κB) activation assessed by NF-κB p65 phosphorylation and IκBα degradation, suppressed LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and intracellular reactive oxygen species (ROS) production. Furthermore, incubation of H9c2 cells with SPRC induced phosphorylation of Akt in a time- and concentration-dependent manner. In addition, SPRC attenuated LPS-induced mRNA and protein expression of tumor necrosis factor-α (TNF-α), and mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS). The effects of SPRC were abolished by cystathionine γ-lyase [CSE-an enzyme that synthesizes hydrogen sulfide (H₂S)] inhibitor, dl-propargylglycine (PAG), SPRC-induced Akt phosphorylation and TNF-α release was also abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Furthermore, SPRC also increased LPS-induced down-regulation expression of CSE and H₂S level in H9c2 cells. PAG abolished SPRC-induced up-regulation of H₂S level. Therefore, we concluded that SPRC produced an anti-inflammatory effect in LPS-stimulated H9c2 cells partly through the CSE/H₂S pathway by impairing IκBα/NF-κB signaling and by activating PI3K/Akt signaling pathway.     

Treatment with TNF-α and IFN-γ alters the activation of SER/THR protein kinases and the metabolic response to IGF-I in mouse c2c12 myogenic cells

The aim of this study was to compare the effects of TNF-α, IL-1β and IFN-γ on the activation of protein kinase B (PKB), p70^S6k, mitogen-activated protein kinase (MAPK) and p90 ^rsk , and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-α abolished this effect. Glucose uptake in cells differentiated in the presence of 10 ng/ml IFN-γ increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I. IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-α nor IFN-γ influenced basal protein synthesis, but both cytokines prevented the IGF-I effect. 10 ng/ml IL-1β did not modify either the basal or IGF-I-dependent glucose uptake and protein synthesis. With the exception of TNF-α causing an 18% decrease in the level of PKB protein, the cellular levels of PKB, p70^S6k, p42^MAPK, p44^MAPK and p90 ^rsk were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal value after 40 min of IGF-I treatment), p42^MAPK (a 2.81-fold increase after 50 min), and the activation of p70^S6k and p90 ^rsk , manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-α or IFN-γ, this IGF-I-mediated PKB and p70^S6k phosphorylation was significantly diminished, and the increase in p42^MAPK and p90 ^rsk phosphorylation was prevented. The basal p42^MAPK phosphorylation in C2C12 cells treated with IFN-γ was high and comparable with the activation of this kinase by IGF-I. Pretreatment of myogenic cells with IL-1β did not modify the IGF-I-stimulated phosphorylation of PKB, p70^S6k, p42^MAPK and p90 ^rsk . In conclusion: i) TNF-α and IFN-γ, but not IL-1β, if present in the extracellular environment during C2C12 myoblast differentiation, prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-α- and IFN-γ-induced IGF-I resistance of protein synthesis could be associated with the decreased phosphorylation of PKB and p70^S6k. iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-γ is PKB independent. iv) The similar effects of TNF-α and IFN-γ on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement of both of these cytokines in protein loss in skeletal muscle.     

Age related changes in T cell mediated immune response and effector memory to Respiratory Syncytial Virus (RSV) in healthy subjects

Respiratory syncytial virus (RSV) is the major pathogen causing respiratory disease in young infants and it is an important cause of serious illness in the elderly since the infection provides limited immune protection against reinfection. In order to explain this phenomenon, we investigated whether healthy adults of different age (20-40; 41-60 and > 60 years), have differences in central and effector memory, RSV-specific CD8+ T cell memory immune response and regulatory T cell expression status. In the peripheral blood of these donors, we were unable to detect any age related difference in term of central (CD45RA^-CCR7⁺) and effector (CD45RA^-CCR7^-) memory T cell frequency. On the contrary, we found a significant increase in immunosuppressive regulatory (CD4⁺25⁺FoxP3⁺) T cells (T_reg) in the elderly. An immunocytofluorimetric RSV pentamer analysis performed on these donors' peripheral blood mononuclear cells (PBMCs), in vitro sensitized against RSV antigen, revealed a marked decline in long-lasting RSV specific CD8+ memory T cell precursors expressing interleukin 7 receptor α (IL-7Rα), in the elderly. This effect was paralleled by a progressive switch from a Th1 (IFN-γ and TNF-α) to a Th2 (IL-10) functional phenotype. On the contrary, an increase in T_reg was observed with aging. The finding of T_reg over-expression status, a prominent Th2 response and an inefficient RSV-specific effector memory CD8+ T cell expansion in older donors could explain the poor protection against RSV reinfection and the increased risk to develop an RSV-related severe illness in this population. Our finding also lays the basis for new therapeutic perspectives that could limit or prevent severe RSV infection in elderly.     

Tumour necrosis factor activates the mitogen-activated protein kinases p38α and ERK in the synovial membrane in vivo

Tumour necrosis factor (TNF) is considered to be a major factor in chronic synovial inflammation and is an inducer of mitogen-activated protein kinase (MAPK) signalling. In the present study we investigated the ability of TNF to activate MAPKs in the synovial membrane in vivo. We studied human TNF transgenic mice – an in vivo model of TNF-induced arthritis – to examine phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38MAPKα in the inflamed joints by means of immunoblot and immunohistochemistry. In addition, the effects of systemic blockade of TNF, IL-1 and receptor activator of nuclear factor-κB (RANK) ligand on the activation of MAPKs were assessed. In vivo, overexpression of TNF induced activation of p38MAPKα and ERK in the synovial membrane, whereas activation of JNK was less pronounced and rarely observed on immunohistochemical analysis. Activated p38MAPKα was predominantly found in synovial macrophages, whereas ERK activation was present in both synovial macrophages and fibroblasts. T and B lymphocytes did not exhibit major activation of any of the three MAPKs. Systemic blockade of TNF reduced activation of p38MAPKα and ERK, whereas inhibition of IL-1 only affected p38MAPKα and blockade of RANK ligand did not result in any decrease in MAPK activation in the synovial membrane. These data indicate that TNF preferentially activates p38MAPKα and ERK in synovial membrane exposed to TNF. This not only suggests that targeted inhibition of p38MAPKα and ERK is a feasible strategy for blocking TNF-mediated effects on joints, but it also shows that even currently available methods to block TNF effectively reduce activation of these two MAPKs.     

An unusual pro-inflammatory role of interleukin-10 induced by arabinosylated lipoarabinomannan in murine peritoneal macrophages

Various species of Mycobacteria produce a major cell wall-associated lipoglycan, called Lipoarabinomannan (LAM), which is involved in the virulence of Mycobacterial species. In this study, we tried to establish the role of the increased IL-10 secretion under Arabinosylated-LAM (Ara-LAM) treatment, the LAM that induces apoptosis in host macrophages or PBMC. We have studied the survival and apoptotic factors by western blotting, and estimated nitrite generation by Griess reaction, quantified iNOS mRNA by semi-quantitative RT-PCR, and ultimately the fate of the cells were studied by Flow Cytometric Analysis of AnnexinV-FITC binding. As per our observations, neutralization of released IL-10 in C57BL/6 peritoneal macrophages prior to Ara-LAM treatment, as well as macrophages from IL-10 knockout (KO) mice treated with Ara-LAM, showed significant down regulation of pro-apoptotic factors and up regulation of survival factors. These effects were strikingly similar to those when peritoneal macrophages were subjected to TNF-α and IL-12 neutralization followed by Ara-LAM-treatment. However, under similar conditions virulent Mannosylated-LAM (from Mycobacterium tuberculosis) treatment of macrophages clearly depicts the importance of IL-10 in the maintenance of pathogenesis, proving its usual immunosuppressive role. Thus, from our detailed investigations we point out an unusual pro-inflammatory action of IL-10 in Ara-LAM treated macrophages, where it behaves in a similar manner as the known Th1 cytokines TNF- α and IL-12. This work is financed by the Council of Scientific and Industrial Research (CSIR), Govt. of India.     

Immunostimulatory effect by aqueous extract of <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> in RAW 264.7 macrophage and whole spleen cells

Hizikia fusiforme is a commonly used food that possesses potent anti-bacterial, anti-fungal, and anti-inflammatory activities. The immunostimulatory activities of aqueous extract of Hizikia fusiforme (HFAE) in RAW 264.7 macrophages and whole spleen cells were investigated. HFAE activated RAW 264.7 macrophages to produce cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a dose-dependent manner. In addition, HFAE induced the mRNA expression of TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages. Moreover, HFAE stimulated proliferation of whole spleen cells and reference mitogen. Taken together, the results demonstrate that HFAE potently activates the immune function by regulating NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage and promoting spleen cell proliferation.     

Cytokine production and lymphocyte proliferation in patients with Nocardia brasiliensis actinomycetoma

IFN-γ, TNF-α, IL-4, IL10 and IL-12 concentrations in the supernatant of peripheral blood mononuclear cell (PBMC) cultures and the in vitro proliferation of PBMC were studied in 25 patients with actinomycetoma caused by Nocardia brasiliensisand in 10 healthy controls from endemic zones. Cell cultures were stimulated by a N. brasiliensiscrude cytoplasmic antigen (NB) and five semi-purified protein fractions (NB2, NB4, NB6, NB8, and NB10) separated by isoelectric. Phytohemagglutinin (PHA) and purified protein derivative (PPD) of Mycobacterium tuberculosiswere used as control antigens. Skin tests were performed by injecting 0.1 ml of candidin and PPD intradermally (ID). Patients showed a poor response to tuberculin, while their response to candidin was more than two fold greater than that observed in the controls. Cell proliferation showed no statistically significant differences in either group. IFN-γ production was higher in the healthy controls than in the patients, whereas TNF-α secretion was slightly higher in the patients’ cultures. IL-4 was detected in the patients’ cultures but not in the controls. IL-10 and IL-12 were present at low concentrations in both groups. These results suggest that patients with actinomycetoma show normal antigen recognition, but with low IFN-γ production, and higher concentrations of IL-4, IL-10 and TNF-α in the patients’ PBMC cultures, indicating that they probably have a Th2 type of immune response.     

Local treatment with the selective IκB kinase β inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-κB is a key regulator of synovial inflammation. We investigated the effect of local NF-κB inhibition in rat adjuvant arthritis (AA), using the specific IκB kinase (IKK)-β blocking NF-κB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-β-induced IκBα phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-α and IL-1-β in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-β-induced TNF-α production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-α-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-κB blockade using a small peptide inhibitor of IKK-β has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-β-targeted NF-κB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.     

Evaluation of Cytokine Production and Phagocytic Activity in Mice Infected with Campylobacter jejuni

The effect of several Campylobacter jejuni strains on the immune response was analyzed in mice after intraperitoneal inoculation with 10¹⁰ colony forming units (CFU). Three C. jejuni strains were assayed: CCUG 6968 (enterotoxigenic), CCUG 7580 (enterotoxigenic), and CCUG 7440 (non-enterotoxigenic). These C. jejuni strains induced a peritoneal inflammatory response and an important increase in the peritoneal phagocyte oxidative activity measured by chemiluminescence assay, as well as an increase in the number of peritoneal cells. Both interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) production by peritoneal cells were not modified. However, C. jejuni 7440 caused a statistically significant increase in TNFα production. These results have demonstrated that different strains of C. jejuni induce an increase of the inflammatory response without a significant cytokine release. However, these infectious microorganisms may be eliminated efficiently by murine macrophages after phagocytosis. Received: 18 February 1999 / Accepted: 6 May 1999     

<Emphasis Type="Italic">Scutellaria</Emphasis> extract and wogonin inhibit tumor-mediated induction of T<Subscript>reg</Subscript> cells via inhibition of TGF-β1 activity

A number of studies have implicated tumor-induced T_reg cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T_reg cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-β1-induced T_reg activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-β1 and T_reg cell frequency as well as peripheral blood TGF-β1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-β1-mediated T_reg activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T_reg culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3β and ERK1/2 signaling by SocL and wogonin in T_reg cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells’ response to TGF-β1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-β1 secretion as well as via inhibition of T cells’ response to TGF-β1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.     

Surface α2-3- and α2-6-sialylation of human monocytes and derived dendritic cells and its influence on endocytosis

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that α2-3-sialylated O-glycans and α2-6- and α2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented α2-6-sialylated N-glycans; ST3Gal1 contributes for the α2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased α2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.     

Infant animal model of pulmonary mycotoxicosis induced by Stachybotrys chartarum

In recent years cases of often fatal pulmonary hemorrhage in infants have been associated with water damaged homes and the toxigenic fungusStachybotrys chartarum. The fungal spores contain mycotoxins which could be injurious to the rapidly developing lung. In order to understand the developmental pathophysiology of this disease we developed an infant rat model of stachybotrytoxicosis describing the effects of fungal spores on survival, growth, histopathology of the lung and respiration. Conidia ofS. chartarum were instilled intratracheally (1.0–8.0 × 10⁵/gm wt.) in 4-dold Sprague-Dawley rat pups. Two control groups received either sterile PBS or a suspension of spores extensively extracted with ethanol to remove toxins. Lethal dose response was determined (LD₅₀ = 2.7 × 10⁵ spores/gm wt.). All dead pups had extensively hemorrhagic lungs. Growth of surviving animals was impaired in a dose-dependent manner. Changes of pulmonary function parameters in rats treated with 1.1 × 10⁵ spores/g were consistent with an increased respiratory resistance. Histology of lungs revealed fresh hemorrhage, sparse hemosiderin-laden macrophages, and evidence of inflammation including thickened alveolar septa infiltrated by lymphocytes and mononuclear cells and intra-alveolar macrophages. Significant increases (p = 0.001) in numbers of macrophages (2-fold), lymphocytes (5-fold) and neutrophils (7-fold) were found in BAL fluid. Hemoglobin was elevated 2-fold (p = 0.004). Proinflammatory mediator IL-1β increased more than 6-fold and TNF-α30-fold (p = 0.001). Extracted spores had a minimal effect on all examined parameters in BAL fluid indicating that mycotoxins are primarily responsible for the hemorrhagic and inflammatory response. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synergy between interleukin-2 and prothymosin α for the increased generation of cytotoxic T lymphocytes against autologous human carcinomas

 Peripheral blood mononuclear cells (PBMC) from cancer patients were cultured in vitro with irradiated autologous tumor cells isolated from malignant effusions (mixed lymphocyte tumor cultures, MLTC) and low-dose (50 IU/ml) recombinant interleukin-2 (IL-2). The combination of IL-2 and prothymosin α (ProTα) resulted in a greater PBMC-induced response to the autologous tumor than that brought about by IL-2 alone. In particular, ProTα specifically enhanced the CD4⁺ T-cell-mediated proliferation against the autologous tumor. CD4⁺ T cells seemed to recognize tumor antigens presented by HLA-DR molecules expressed on the autologous monocytes, since preincubation of the latter with an anti-HLA-DR monoclonal antibody (mAb) abrogated the response. In addition, MLTC set up with IL-2 and ProTα also generated more MHC-class-I-restricted cytotoxic T lymphocytes (CTL) against the autologous tumor than did MLTC set up with IL-2 alone. The MLTC-induced CTL contained high levels of cytoplasmic perforin and their development was strictly dependent on the presence of both autologous CD4⁺ T cells and monocytes. In the absence of either population there was a strong impairment of both proliferative and cytotoxic responses which was not restored by the presence of ProTα. In contrast, when both cell populations were present, ProTα exerted optimal enhancement of CD4⁺ T cell proliferation, which was associated with potentiated CTL responses. Our data emphasize the role of ProTα for the enhancement of IL-2-induced CTL responses against autologous tumor cells. Such responses require collaborative interactions between CD4⁺, CD8⁺ T cells and monocytes as antigen-presenting cells. Our data are relevant for adoptive immunotherapeutic settings utilizing IL-2 and ProTα-induced autologous-tumor-specific CTL. Received: 2 March 2000 / Accepted: 1 June 2000     

Effect of histidine methylation on the recognition of OVA323-336 T-epitope: Synthesis of N-α-Fmoc-N-τ-methyl-L-histidine and its use for the synthesis of two histidine methylated analogues of OVA323-336

Summary N-α-Fmoc-N-τ-methyl-L-histidine was prepared in three steps fromN-α-Boc-L-histidine by treatment with methyliodine in DMF at−10°C, deprotection of theN-α position in pure TFA and subsequent reprotection by Fmoc-chloroformate in a 5% Na₂CO₃/dioxane mixture.N-α-Fmoc-N-τ-methyl-L-histidine was then used for the solid-phase synthesis of two analogues of the OVA_323–336 T-epitope, methylated on His₃₃₁ and on His_328/331, respectively. These peptides were tested for their ability to activate 3 DO-54.8 T-cell hybridoma when presented by fixed A-20.1.11 antigen presenting cells, and no significant difference was observed in IL-2 production.     

Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion

Mast cell (MC) activation in the rheumatoid lesion provides numerous mediators that contribute to inflammatory and degradative processes, especially at sites of cartilage erosion. MC activation in rheumatoid synovial tissue has often been associated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β production by adjacent cell types. By contrast, our in situ and in vitro studies have shown that the production of IL-15 was independent of MC activation, and was not related to TNF-α and IL-1β expression. Primary cultures of dissociated rheumatoid synovial cells produced all three proinflammatory cytokines, with production of IL-1β exceeding that of TNF-α, which in turn exceeded that of IL-15. In vitro cultures of synovial macrophages, synovial fibroblasts and articular chondrocytes all produced detectable amounts of free IL-15, macrophages being the most effective.