Individual immune cell and cytokine profiles determine platelet-rich plasma composition.

OBJECTIVE: Platelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes. DESIGN: For each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed. RESULTS: All three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content. CONCLUSIONS: Individuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual's immune profile and the concentration method appear to impact the final PRP product. TRIAL REGISTRATION: This study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

Impaired response of blood neutrophils to cell-death stimulus differentiates AQP4-IgG-seropositive NMOSD from MOGAD.

BACKGROUND: In neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), neutrophils are found in CNS lesions. We previously demonstrated that NMOSD neutrophils show functional deficiencies. Thus, we hypothesized that neutrophil accumulation in the CNS may be facilitated by impairments affecting mechanisms of neutrophil death. OBJECTIVE: To evaluate cell death in blood neutrophils from aquaporin-4 (AQP4)-IgG-seropositive NMOSD and MOGAD patients as well as matched healthy controls (HC) using in vitro assays. METHODS: Twenty-eight AQP4 + NMOSD and 19 MOGAD patients in stable disease phase as well as 45 age- and sex-matched HC were prospectively recruited. To induce cell death, isolated neutrophils were cultured with/without phorbol 12-myristate 13-acetate (PMA). Spontaneous and PMA-induced NETosis and apoptosis were analyzed using 7-AAD and annexin-V by flow cytometry. Caspase-3 was assessed by western blot. Myeloperoxidase-DNA complexes (MPO-DNA), MPO and elastase were evaluated by ELISA, and cell-free DNA (cfDNA) by a fluorescence-based assay. Reactive oxygen species (ROS) were evaluated by a dihydrorhodamine 123-based cytometric assay. Serum GM-CSF, IL-6, IL-8, IL-15, TNF-ɑ and IL-10 were evaluated by multiplex assays, and neurofilament light chain (NfL) by single-molecule array assay. RESULTS: In response to PMA, neutrophils from AQP4 + NMOSD but not from MOGAD patients showed an increased survival, and subsequent reduced cell death (29.6% annexin V+ 7-AAD+) when compared to HC (44.7%, p = 0.0006). However, AQP4 + NMOSD also showed a mild increase in annexin V+ 7-AAD- early apoptotic neutrophils (24.5%) compared to HC (20.8%, p = 0.048). PMA-induced reduction of caspase-3 activation was more pronounced in HC (p = 0.020) than in AQP4 + NMOSD neutrophils (p = 0.052). No differences were observed in neutrophil-derived MPO-DNA or serum levels of MPO, elastase, IL-6, IL-8 and TNF-ɑ. IL-15 levels were increased in both groups of patients. In AQP4 + NMOSD, an increase in cfDNA, GM-CSF and IL-10 was found in serum. A positive correlation among cfDNA and NfL was found in AQP4 + NMOSD. CONCLUSIONS: AQP4 + NMOSD neutrophils showed an increased survival capacity in response to PMA when compared to matched HC neutrophils. Although the data indicate that the apoptotic but not the NETotic response is altered in these neutrophils, additional evaluations are required to validate this observation.

Generation of HBsAg-reactive T- and B-cells following HBV vaccination in serological non-responders under hemodialysis treatment.

HBV vaccination is recommend for hemodialysis patients, but only 50-60% of the patients show seroconversion. HBV vaccine-induced generation of HBV reactive T and B cells could be detected regardless of their capacity to mount a serological response, indicating that patients without seroconversion are potentially protected by their HBV-reactive T cell pool.

Killer-like receptors and GPR56 progressive expression defines cytokine production of human CD4+ memory T cells.

All memory T cells mount an accelerated response on antigen reencounter, but significant functional heterogeneity is present within the respective memory T-cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, including KLRB1, KLRG1, GPR56, and KLRF1, help define low, high, or exhausted cytokine producers within human peripheral and intrahepatic CD4+ memory T-cell populations. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1+KLRG1+GPR56+ CD4 T cells. By contrast, KLRF1 expression is associated with T-cell exhaustion and reduced TNF/IFN-γ production. Lastly, TCRß repertoire analysis and in vitro differentiation support a regulated, progressive expression for these markers during CD4+ memory T-cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development.

Treatment of advanced gastrointestinal cancer with genetically modified autologous mesenchymal stem cells: Results from the phase 1/2 TREAT-ME-1 trial.

TREAT-ME-1, a Phase 1/2 open-label multicenter, first-in-human, first-in-class trial, evaluated the safety, tolerability and efficacy of treatment with genetically modified autologous mesenchymal stromal cells (MSC), MSC_ apceth_101, in combination with ganciclovir in patients with advanced gastrointestinal adenocarcinoma. Immunological and inflammatory markers were also assessed. All patients (3 in Phase 1; 7 in Phase 2) received three treatment cycles of MSC_apceth_101 at one dose level on Day 0, 7, and 14 followed by ganciclovir administration according to the manufacturer's instructions for 48─72 h after MSC_apceth_101 injection. Ten patients were treated with a total dose of 3.0 x 106 cells/kg MSC_apceth_101. 36 adverse events and six serious adverse events were reported. Five patients achieved stable disease (change in target lesions of -2 to +28%). For all patients, the median time to progression was 1.8 months (95% CI: 0.5, 3.9 months). Median overall survival could not be estimated as 8/10 patients were still alive at the end of the study (1 year) and therefore censored. Post-study observation of patients showed a median overall survival of 15.6 months (ranging from 2.2─27.0 months). Treatment with MSC_apceth_101 and ganciclovir did not induce a consistent increase or decrease in levels of any of the tumor markers analyzed. No clear trends in the immunological markers assessed were observed. MSC_apceth_101 in combination with ganciclovir was safe and tolerable in patients with advanced gastrointestinal adenocarcinoma, with preliminary signs of efficacy in terms of clinical stabilization of disease.

Gravitational stress during parabolic flights reduces the number of circulating innate and adaptive leukocyte subsets in human blood.

Gravitational stress occurs during space flights or certain physical activities including extreme sports, where the change in experienced gravitational acceleration can reach large magnitudes. These changes include reduction and increase in the physical forces experienced by the body and may potentially induce pathogenic alterations of physiological processes. The immune system is known to regulate most functions in the human organism and previous studies suggest an impairment of the immune function under gravitational stress. However, systematic studies aiming to investigate the effect of gravitational stress on cellular immune response in humans are lacking. Since parabolic flights are considered as feasible model to investigate a short-term impact of gravitational changes, we evaluated the influence of gravitational stress on the immune system by analyzing leukocyte numbers before and after parabolic flight maneuvers in human blood. To correct for circadian effects, samples were taken at the corresponding time points on ground the day before the flight. The parabolic flight maneuvers led to changes in numbers of different leukocyte subsets. Naïve and memory T and B cell subsets decreased under gravitational stress and lower numbers of basophils and eosinophils were observed. Only circulating neutrophils increased during the parabolic flight. The observed changes could not be attributed to stress-induced cortisol effects, since cortisol levels were not affected. Our data demonstrate that the gravitational stress by parabolic flights can affect all parts of the human immune system. Consequently, it is possible that gravitational stress can have clinically relevant impacts on the control of immune responses.

Treatment of advanced gastrointestinal cancer with genetically modified autologous mesenchymal stem cells - TREAT-ME-1 - a phase I, first in human, first in class trial.

PURPOSE: This phase I, first in human, first in class clinical study aimed at evaluating the safety, tolerability and efficacy of treatment with genetically modified mesenchymal stromal cells (MSC) in combination with ganciclovir (GCV). MSC_apceth_101 are genetically modified autologous MSCs used as vehicles for a cell-based gene therapy in patients with advanced gastrointestinal adenocarcinoma. EXPERIMENTAL DESIGN: The study design consisted of a dose-escalation 3 + 3 design. All patients (n = 6) were treated with up to three applications of MSC_apceth_101, followed by GCV infusions given on three consecutive days starting 48 hours after injection of MSC_apceth_101. Three of six patients received a total dose of 1.5 × 106 cells/kg. Two patients received three doses of 1 × 106 cells/kg, while one patient received only two doses of 1 × 106 cells/kg due to a SADR. RESULTS: Six patients received MSC_apceth_101. No IMP-related serious adverse events occurred. Adverse-events related to IMP-injection were increased creatinine, cough, fever, and night sweat. TNF, IL-6, IL-8, IL-10 and sE-Selectin, showed that repeated application is immunologically safe, but induces a switch of the functional properties of monocytes to an inflammatory phenotype. Treatment induced stable disease in 4/6 patients, and progressive disease in 2/6 patients. CONCLUSION: Treatment with MSC_apceth_101 in combination with GCV demonstrated acceptable safety and tolerability in patients with advanced gastrointestinal adenocarcinoma.

TCAIM decreases T cell priming capacity of dendritic cells by inhibiting TLR-induced Ca2+ influx and IL-2 production.

We previously showed that the T cell activation inhibitor, mitochondrial (Tcaim) is highly expressed in grafts of tolerance-developing transplant recipients and that the encoded protein is localized within mitochondria. In this study, we show that CD11c(+) dendritic cells (DCs), as main producers of TCAIM, downregulate Tcaim expression after LPS stimulation or in vivo alloantigen challenge. LPS-stimulated TCAIM-overexpressing bone marrow-derived DC (BMDCs) have a reduced capacity to induce proliferation of and cytokine expression by cocultured allogeneic T cells; this is not due to diminished upregulation of MHC or costimulatory molecules. Transcriptional profiling also revealed normal LPS-mediated upregulation of the majority of genes involved in TLR signaling. However, TCAIM BMDCs did not induce Il2 mRNA expression upon LPS stimulation in comparison with Control-BMDCs. In addition, TCAIM overexpression abolished LPS-mediated Ca(2+) influx and mitochondrial reactive oxygen species formation. Addition of IL-2 to BMDC-T cell cocultures restored the priming capacity of TCAIM BMDCs for cocultured allogeneic CD8(+) T cells. Furthermore, BMDCs of IL-2-deficient mice showed similarly abolished LPS-induced T cell priming as TCAIM-overexpressing wild type BMDCs. Thus, TCAIM interferes with TLR4 signaling in BMDCs and subsequently impairs their T cell priming capacity, which supports its role for tolerance induction.

Association of TLR3-hyporesponsiveness and functional TLR3 L412F polymorphism with recurrent herpes labialis.

HSV-1 persistently infects almost 90% of our population; however, only 30% of the infected subjects suffer from recurrent herpes lesions, most frequently herpes labialis (HL). We hypothesized that variations in toll-like receptor (TLR) functions might contribute to HL susceptibility. In our study, the TLR-2/1,-3, and -7/8 responses of immune cell subsets derived from asymptomatic HSV-1 carriers were compared with responses of subjects with HL history. Remarkably, natural killer (NK) cells isolated from HL subjects showed significantly lower IFN-γ responses selectively to the TLR3 agonist poly(I:C). Furthermore, the TLR3 L412F genetic polymorphism was found to reduce NK cell TLR3-responsiveness and is associated with susceptibility to recurrent HL. The TLR3 response detected in HL total peripheral blood mononuclear cells (PBMCs), however, was not impaired, indicating restoration of NK cell TLR3-deficiency through co-stimulatory functions. In conclusion, our results suggest that decreased TLR3 response of NK cells is associated with HL susceptibility; and potentially explain why symptomatic outbreak of HL usually occurs after stress or prolonged UV light exposure, when host co-stimulatory functions are disturbed.

Biology of interleukin-10.

Interleukin (IL)-10 is the most important cytokine with anti-inflammatory properties besides TGF-ß and IL-35. It is produced by activated immune cells, in particular monocytes/macrophages and T cell subsets including Tr1, Treg, and Th1 cells. IL-10 acts through a transmembrane receptor complex, which is composed of IL-10R1 and IL-10R2, and regulates the functions of many different immune cells. In monocytes/macrophages, IL-10 diminishes the production of inflammatory mediators and inhibits antigen presentation, although it enhances their uptake of antigens. Additionally, IL-10 plays an important role in the biology of B cells and T cells. The special physiological relevance of this cytokine lies in the prevention and limitation of over-whelming specific and unspecific immune reactions and, in consequence, of tissue damage. At the same time, IL-10 strengthens the "scavenger"-function and contributes to induced tolerance. This review provides an overview about the cellular sources, molecular mechanisms, effects, and biological role of IL-10.

Deletion of the RNA-binding proteins ZFP36L1 and ZFP36L2 leads to perturbed thymic development and T lymphoblastic leukemia.

ZFP36L1 and ZFP36L2 are RNA-binding proteins (RBPs) that interact with AU-rich elements in the 3' untranslated region of mRNA, which leads to mRNA degradation and translational repression. Here we show that mice that lacked ZFP36L1 and ZFP36L2 during thymopoiesis developed a T cell acute lymphoblastic leukemia (T-ALL) dependent on the oncogenic transcription factor Notch1. Before the onset of T-ALL, thymic development was perturbed, with accumulation of cells that had passed through the beta-selection checkpoint without first expressing the T cell antigen receptor beta-chain (TCRbeta). Notch1 expression was higher in untransformed thymocytes in the absence of ZFP36L1 and ZFP36L2. Both RBPs interacted with evolutionarily conserved AU-rich elements in the 3' untranslated region of Notch1 and suppressed its expression. Our data establish a role for ZFP36L1 and ZFP36L2 during thymocyte development and in the prevention of malignant transformation.

The role of mitogen-activated protein kinase-activated protein kinase 2 in the p38/TNF-alpha pathway of systemic and cutaneous inflammation.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream molecule of p38, involved in the production of TNF-alpha, a key cytokine, and an established drug target for many inflammatory diseases. We investigated the role of MK2 in skin inflammation to determine its drug target potential. MK2 deficiency significantly decreased plasma TNF-alpha levels after systemic endotoxin application. Deficient mice showed decreased skin edema formation in chronic 2-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritative dermatitis and in subacute 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. Surprisingly, MK2 deficiency did not inhibit edema formation in subacute 2,4-dinitrochlorobenzene (DNCB)-induced contact allergy and even increased TNF-alpha and IL-1beta levels as well as granulocyte infiltration in diseased ears. Ear inflammation in this model, however, was inhibited by TNF-alpha neutralization as it was in the subacute DNFB model. MK2 deficiency also did not show anti-inflammatory effects in acute DNFB-induced contact hypersensitivity, whereas the p38 inhibitor, SB203580, ameliorated skin inflammation supporting a pathophysiological role of p38. When evaluating possible mechanisms, we found that TNF-alpha production in MK2-deficient spleen cells was strongly diminished after TLR stimulation but less affected after T-cell receptor stimulation. Our data suggest that MK2, in contrast to its downstream effector molecule, TNF-alpha, has a rather elusive role in T-cell-dependent cutaneous inflammation.

Expression of tolerance associated gene-1, a mitochondrial protein inhibiting T cell activation, can be used to predict response to immune modulating therapies.

Immune modulating therapies gain increasing importance in treatment of patients with autoimmune diseases such as psoriasis. None of the currently applied biologics achieves significant clinical improvement in all treated patients. Because the therapy with biologics is cost intensive and sometimes associated with side effects, noninvasive diagnostic tools for early prediction of responders are of major interest. We studied the effects of Alefacept (LFA3Ig), an approved drug for treatment of psoriasis, on leukocytes in vitro and in vivo to identify gene markers predictive for treatment response and to further investigate its molecular mechanisms of action. In an open-label study, 20 psoriasis patients were treated weekly with 15 mg Alefacept over 12 wk. We demonstrate that transcription of the tolerance-associated gene (TOAG-1) is significantly up-regulated whereas receptor for hyaluronic acid mediated migration (RHAMM) transcription is down-regulated in PBMCs of responding patients before clinical improvement. TOAG-1 is exclusively localized within mitochondria. Overexpression of TOAG-1 in murine T cells leads to increased susceptibility to apoptosis. Addition of Alefacept to stimulated human T cells in vitro resulted in reduced frequencies of activated CD137(+) cells, increased TOAG-1 but reduced RHAMM expression. This was accompanied by reduced proliferation and enhanced apoptosis. Inhibition of proliferation was dependent on enhanced PDL1 expression of APCs. Thus, peripheral changes of TOAG-1 and RHAMM expression can be used to predict clinical response to Alefacept treatment in psoriasis patients. In the presence of APCs Alefacept can inhibit T cell activation and survival by increasing expression of TOAG-1 on T cells and PDL1 on APCs.

Sustained BK viruria as an early marker for the development of BKV-associated nephropathy: analysis of 4128 urine and serum samples.

BACKGROUND: BKV reactivation plays the causative role in the development of BKV-associated nephropathy (BKVAN). Because of the lack of effective therapy, early diagnosis of BKV reactivation is paramount for the prevention of BKVAN. Resting in uroepithelial cells, BKV is excreted first in urine before it can be detected in plasma. The present study analyzed predictive value of BK viruria for the development of BK viremia and its possible advantage for the early BKVAN prediction. METHODS: Total of 4128 urine and serum samples obtained from renal transplant patients were analyzed for BKV positivity by real-time polymerase chain reaction in 433 patients in cross-sectional and in 233 patients in longitudinal manner, respectively. The prospective longitudinal analysis included seven measurements during the first posttransplant year. RESULTS: A total of 7% and 19% patients were positive for BKV in serum and urine, respectively. Sustained BK viruria showed sensitivity of 100% and specificity of 94% for BK viremia and was associated with significantly higher level of BK load than the patients with transient viruria (P<0.01). Interestingly, BK viremia was preceded by BK viruria: the peak of viral load and number of positive patients appeared during the third and fifth posttransplant month for urine and serum, respectively. BKVAN diagnosed in 21.4% of patient with persistent BK viruria appeared 5 and 11 weeks after BKV reactivation in serum and urine, respectively, was detected. CONCLUSION: Sustained BK viruria is a reliable marker allowing an early identification of patients at high risk of BKVAN development and therefore assure precocious therapeutic interventions.

IL-10 protects monocytes and macrophages from complement-mediated lysis.

Phagocytes, such as monocytes and macrophages, are important cells of the innate immunity in the defense against microbes. So far, it is unclear how these cells survive at the site of combat against microbes, where a hostile inflammatory environment prevails with strong complement activity. We hypothesized that IL-10, a key cytokine involved in the resolution of inflammation, induces resistance to complement attack. Here, we demonstrate for the first time such a cell-protective effect of IL-10 on human monocytes and macrophages. IL-10 is indeed able to protect these cell types in an in vitro model of complement lysis triggered by an anti-MHCI antibody or by binding of zymosan. Investigating potential underlying mechanisms, we found that IL-10 up-regulated the expression of complement regulatory membrane protein CD59 and the general cell-protective stress protein HO-1 in human monocytes. However, further functional analysis failed to link these individual IL-10-mediated effects with the increased protection from complement lysis. Blocking the protective effect of CD59 with an antibody increased complement lysis but did not abrogate the IL-10-protective effect. Interestingly, chemical interference with HO-1 activity did abrogate the protective effect of IL-10, but siRNA-mediated knockdown of HO-1 did not confirm this observation. Our results suggest that IL-10 generates pathogen-clearing phagocytes, which are resistant to complement lysis and thereby, enabled to survive longer in a hostile inflammatory environment.

IL-10 interferes directly with TCR-induced IFN-gamma but not IL-17 production in memory T cells.

IL-10 is a potent immunoregulatory and anti-inflammatory cytokine. However, therapeutic trials in chronic inflammation have been largely disappointing. It is well established that IL-10 can inhibit Th1 and Th2 cytokine production via indirect effects on APC. Less data are available about the influence of IL-10 on IL-17 production, a cytokine which has been recently linked to chronic inflammation. Furthermore, there are only few reports about a direct effect of IL-10 on T cells. We demonstrate here that IL-10 can directly interfere with TCR-induced IFN-gamma production in freshly isolated memory T cells in the absence of APC. This effect was independent of the previously described effects of IL-10 on T cells, namely inhibition of IL-2 production and inhibition of CD28 signaling. In contrast, IL-10 did not affect anti-CD3/anti-CD28-induced IL-17 production from memory T cells even in the presence of APC. This might have implications for the interpretation of therapeutic trials in patients with chronic inflammation where Th17 cells contribute to pathogenesis.

A critical control element for interleukin-4 memory expression in T helper lymphocytes.

Naive T helper (Th) lymphocytes are induced to express the il4 (interleukin-4) gene by simultaneous signaling through the T cell receptor and the interleukin (IL)-4 receptor. Upon restimulation with antigen, such preactivated Th lymphocytes can reexpress the il4 gene independent of IL-4 receptor signaling. This memory for expression of the il4 gene depends on epigenetic modification of the il4 gene locus and an increased expression of GATA-3, the key transcription factor for Th2 differentiation. Here, we have identified a phylogenetically conserved sequence, the conserved intronic regulatory element, in the first intron of the il4 gene containing a tandem GATA-3 binding site. We show that GATA-3 binds to this sequence in a position- and orientation-dependent manner, in vitro and in vivo. DNA demethylation and histone acetylation of this region occurs early and selectively in differentiating, IL-4-secreting Th2 lymphocytes. Deletion of the conserved element by replacement of the first exon and part of the first intron of the il4 gene with gfp leads to a defect in the establishment of memory for expression of IL-4, in that reexpression of IL-4 still requires costimulation by exogenous IL-4. The conserved intronic regulatory element thus links the initial epigenetic modification of the il4 gene to GATA-3 and serves as a genetic control element for memory expression of IL-4.

New insights into the molecular mechanism of interleukin-10-mediated immunosuppression.

Interleukin-10 (IL-10) is an important immunomodulatory cytokine, which has attracted much attention because of its anti-inflammatory properties. It reduces antigen presentation and inhibits T cell activation. IL-10-treated myeloid cells lose their ability to respond toward the endotoxin lipopolysaccharide (LPS) with the production of several proinflammatory mediators. Thereby, IL-10 limits excessive inflammatory reactions in response to endotoxin as it occurs in colitis or endotoxin shock. Mice can be tolerized toward endotoxin shock when pretreated with a sublethal dose of LPS. This can be mimicked in vitro as LPS desensitization, resulting in a similar LPS hyporesponsiveness as observed with IL-10 pretreatment. However, an early block in LPS signaling characterizes LPS desensitization, whereas IL-10 seems to target late events. Controversial reports have been published where IL-10 would interfere with the induction of proinflammatory mediators, and little is known about the molecular mechanisms behind the anti-inflammatory activities of IL-10. Some recent publications have tried to gain more insight into the molecular mechanism of IL-10 by gene-expression profiling and functional studies in myeloid-derived cells. These results are reviewed here and compared with the progress that has been made to understand the induction of endotoxin tolerance by LPS itself.

Expression profiling of IL-10-regulated genes in human monocytes and peripheral blood mononuclear cells from psoriatic patients during IL-10 therapy.

Interleukin-10 (IL-10), originally identified as an inhibitor of pro-inflammatory cytokine production, exerts multiple immunomodulatory functions. Its ability to inhibit a Th1 response has been used in clinical trials for the treatment of inflammatory diseases including psoriasis. However, little is known about the molecular mechanisms of IL-10 functions. We aimed at identifying possible mediators of in vitro IL-10 treatment in monocytes by gene chip technology using Hu95a Affymetrix mRNA arrays with 12,000 genes. To prove relevance of the identified genes for the clinical situation we compared these in vitro results with genes being regulated by IL-10 in peripheral blood mononuclear cells from psoriatic patients undergoing IL-10 therapy. A high proportion of the 1,600 genes up-regulated and 1,300 genes down-regulated in vitro was found to be similarly regulated in vivo. Some genes, which were previously unknown to be regulated by IL-10, can be assigned to known IL-10 functions like e.g. the increase of pathogen clearance. Other new potentially immunomodulating genes have been identified to be regulated by IL-10, but their impact needs to be experimentally evaluated. We could confirm a recently reported up-regulation of heme oxygenase-1 (HO-1). However, we demonstrate that the anti-inflammatory mechanisms of IL-10 remain functional even when HO-1 is irreversibly inhibited.

IFN-gamma regulation in anti-CD4 antibody-induced T cell unresponsiveness.

The anti-rat CD4 mAb RIB5/2 is very potent in inducing allospecific tolerance in vivo. It is interesting that the unresponsiveness is breakable by exogenous IL-2 applied during the induction phase of tolerance. The molecular mechanisms underlying anti-CD4 antibody-mediated inhibition of allospecific T cell activation and how this is antagonized by exogenous IL-2 were investigated. Anti-CD4 treatment, in vivo and in vitro, completely abrogated IL-2 production by alloreactive T cells. In contrast, anti-CD4-treated alloactivated T cells showed similar IFN-gamma mRNA expression as untreated alloactivated T cells but did not secrete any protein. Thus, the anti-CD4 antibody cannot prevent IFN-gamma mRNA expression but is interfering with posttranscriptional mechanisms that control IFN-gamma production during alloactivation of T cells. Addition of IL-2 but not IL-15 to anti-CD4-treated alloactivated T cells restored IFN-gamma protein production without leading to enhanced IFN-gamma mRNA expression. Further investigations revealed a diminished activation of translation initiation factor eIF2alpha in anti-CD4-treated T cells, which was restored by exogenous IL-2. As activated eIF2alpha is essential for the translation of IFN-gamma mRNA, the results may explain the reversibility of anti-CD4-induced unresponsiveness by exogenous IL-2. Furthermore, these results not only shed further light onto the molecular mechanisms of tolerance induction but also reveal the possible weaknesses of anti-CD4 antibody-induced unresponsiveness.