InVivoMAb anti-human IL-10

Catalog #BE0441
Clone:
JES3-19F1
Reactivities:
Human

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Product Details

The JES3-19F1 monoclonal antibody reacts with human interleukin 10 (hIL-10) and its viral homolog, Epstein-Barr virus (EBV) IL-10 (ebvIL-10 or vIL-10). IL-10 is synthesized by a variety of cells, including T-cells, macrophages, and mast cells. IL-10 is a pleiotropic cytokine essential to the regulation of inflammatory and immune responses, e.g., suppression of proinflammatory cytokine production by monocytes and neutrophils, and macrophage as well as T cell effector functions. IL-10 interacts with its receptor leading to a JAK1 and STAT2/STAT3-mediated anti-inflammatory response. IL-10 targets macrophages and monocytes, thereby inhibiting their release of pro-inflammatory cytokines, including GM-CSF, G-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, and TNF-alpha. Several in vitro studies have shown that the JES3-19F1 antibody neutralizes endogenous and recombinant human IL-10 and vIL-10. Notably, this antibody does not detect murine IL-10. The JES3-19F1 antibody is useful as a capture or coating antibody in ELISA applications, wherein it has been documented to be compatible with the biotinylated anti-human IL10 clone JES3-12G8 detection antibody. The JES3-12G8 antibody has been shown to be sufficient to reduce bacterial load in Mycobacterium avium infected hu10Tg/muIL-10−/− mice.

Specifications

Isotype Rat IgG2a, κ
Recommended Isotype Control(s) InVivoMAb rat IgG2a isotype control, anti-trinitrophenol
Recommended Dilution Buffer InVivoPure pH 7.0 Dilution Buffer
Immunogen Epstein-Barr virus IL-10 (vIL-10)
Reported Applications in vivo neutralization of IL-10
in vitro neutralization of IL-10
Immunohistochemistry (frozen)
Immunohistochemistry (paraffin)
Functional assays
Flow cytometry
Western blot
ELISA
Formulation PBS, pH 7.0
Contains no stabilizers or preservatives
Endotoxin <2EU/mg (<0.002EU/μg)
Determined by LAL gel clotting assay
Purity >95%
Determined by SDS-PAGE
Sterility 0.2 µm filtration
Production Purified from cell culture supernatant in an animal-free facility
Purification Protein G
Molecular Weight 150 kDa
Storage The antibody solution should be stored at the stock concentration at 4°C. Do not freeze.
Flow Cytometry
Mion F, Martinis E, Pucillo CEM, Tonon S. (2021). "Purification of Murine and Human IL-10-Producing B Cells from Different Anatomical Compartments" Methods Mol Biol . PubMed

IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).

Immunohistochemistry (paraffin)
Rai V, Agrawal DK. (2021). "Immunomodulation of IL-33 and IL-37 with Vitamin D in the Neointima of Coronary Artery: A Comparative Study between Balloon Angioplasty and Stent in Hyperlipidemic Microswine" Int J Mol Sci 22(16):8824. PubMed

Inflammation is a major contributor to the development and progression of atherosclerosis. Interleukin (IL)-33 and IL-37, members of the IL-1 family, modulate inflammation, with IL-33 having a pro-inflammatory effect and IL-37 having anti-inflammatory properties. IL-37 is constitutively expressed at low levels but upregulated in inflammatory contexts. The aim of this study was to evaluate the effect of vitamin D on the expression of IL-33, IL-37, macrophages, and caspase-1 in the neointimal tissue of coronary artery in Yucatan microswine with vitamin D deficient, sufficient, and supplemented status. The intimal injury was induced by balloon angioplasty and stenting in the coronary artery, and tissues were harvested after 6 months. The expression of various proteins of interest was evaluated by immunostaining. Increased expression of IL-33 and IL-37 in the neointimal tissue of the vitamin D deficient, as compared to the sufficient and supplemented microswine, as revealed by histological evaluation and semi-quantitative analysis, suggested the immunomodulatory effect of vitamin D on the expression of IL-33 and IL-37. The minimal expression or absence of IL-33 and IL-37 expression in stented arteries is suggestive of an attenuated inflammatory response in stented arteries, compared to balloon angioplasty. The decreased IL-33 expression in the sufficient and supplemented microswine could be a potential mechanism for controlling the inflammatory process and neointima formation leading to attenuated luminal narrowing of the coronary artery. Overall, these results support supplementation of vitamin D to attenuate inflammation, neointima formation, and restenosis.

Immunohistochemistry (paraffin)
Sá MC, de Matos FR, Conceição TS, Leitão AC, Freitas RA. (2017). "Immunoexpression of tumour necrosis factor-α, interleukin-1α and interleukin-10 on odontogenic cysts and tumours" Int Endod J 50(5):437-445. PubMed

Aim: To analyse the immunoreactivity of IL-1α, TNF-α and IL-10 in odontogenic cysts and tumours and to investigate possible associations with established biological behaviours of these different lesions. Methodology: Immunohistochemical expression of anti-IL-1α, anti-TNF-α and anti-IL-10 antibodies was assessed on epithelium and mesenchyme of 20 radicular cysts (RCs), 20 residual cysts (RECs), 20 dentigerous cysts (DCs), 18 solid ameloblastomas (SAs), 20 keratocystic odontogenic tumours (KCOTs) and 15 dental follicles (DFs). Comparative analysis of data was performed using the nonparametric Wilcoxon signed-rank test and Kruskal-Wallis's test. Results: Significantly greater expression of IL-1α in the epithelium was noted in RC, KCOT and SA (P = 0.01), whilst IL-10 and TNF-α was in the epithelium of RC, DC and KCOT (P < 0.01). In the mesenchyme, significantly greater immunopositivity was observed for IL-1α, IL-10 and TNF-α in KCOT, DC and RC (P < 0.01). In epithelial and mesenchymal tissues, there were a significant number of cases of RC and DC with IL-1α < IL-10 ratio (P < 0.01), whilst SA and KCOT showed IL-1α > IL-10 (P < 0.01). There was a significantly greater percentage of DF, DC and KCOT with TNF-α > IL10 ratio (P < 0.01). Conclusion: These results suggest involvement of the proteins in the pathogenesis of odontogenic cysts and tumours, with emphasis on the highest immunoreactivity of osteolysis stimulating factors in tumours with aggressive biological behaviour, such as SA and KCOT.

in vitro neutralization of IL-10, Western Blot, ELISA
Brodeur ND, Spencer JV. (2010). "Antibodies to human IL-10 neutralize ebvIL-10-mediated cytokine suppression but have no effect on cmvIL-10 activity" Virus Res 153(2):265-8. PubMed

Interleukin-10 is a pivotal determinant of virus clearance or persistence. Two human herpesviruses, Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) are unique among persistent viruses because they not only trigger production of host IL-10, but both viruses also encode homologs of IL-10 that are expressed during infection. Because anti-human IL-10 antibodies have diagnostic value and therapeutic potential for many chronic infections, cross-reactivity with ebvIL-10 and cmvIL-10 was evaluated in this study. Six of seven anti-hIL-10 antibodies tested recognized ebvIL-10 and neutralized its immunosuppressive activity. In contrast, cmvIL-10 was neither recognized nor neutralized by any anti-human IL-10 antibody. These findings demonstrate that IL-10-neutralizing treatments in HCMV- or EBV-infected patients may require consideration of the contribution of viral IL-10 to disease pathology.

ELISA
Stefura WP, Campbell JD, Douville R, Stinson MJ, Simons FE, Becker AB, HayGlass KT. (2008). "Ultrasensitive ELISA for measurement of human cytokine responses in primary culture" Methods Mol Med . PubMed

ELISAs offer excellent specificity and, once fully optimized, sensitivity that rivals that of bioassays. The major variables that need to be experimentally determined when developing an ELISA are the optimal number of fresh cells required per well, the optimal antigen concentrations for stimulation, period of culture, and the anticipated intensity of the response. In this chapter, we review the major factors to be considered in the development and application of ultrasensitive ELISAs to the analysis of human immune responses. We specify the conditions we have found to be optimal for quantifying a number of cytokines of demonstrated relevance to human immune regulation and discuss the major pitfalls inherent in this approach.

Functional Assays
Morandi F, Levreri I, Bocca P, Galleni B, Raffaghello L, Ferrone S, Prigione I, Pistoia V. (2007). "Human neuroblastoma cells trigger an immunosuppressive program in monocytes by stimulating soluble HLA-G release" Cancer Res 67(13):6433-41. PubMed

HLA-G is overexpressed in different tumors and plays a role in immune escape. Because no information is available on HLA-G in relation to human neuroblastoma, we have investigated the expression of membrane-bound and secretion of soluble isoforms of HLA-G in neuroblastoma and functionally characterized their immunosuppressive activities. At diagnosis, serum soluble HLA-G (sHLA-G) levels were significantly higher in patients than in age-matched healthy subjects. In addition, patients who subsequently relapsed exhibited higher sHLA-G levels than those who remained in remission. Neuroblastoma patient sera selected according to high sHLA-G concentrations inhibited natural killer (NK) cell and CTL-mediated neuroblastoma cell lysis. Such lysis was partially restored by serum depletion of sHLA-G. In 6 of 12 human neuroblastoma cell lines, low HLA-G surface expression was not up-regulated by IFN-gamma. Only the ACN cell line secreted constitutively sHLA-G. IFN-gamma induced de novo sHLA-G secretion by LAN-5 and SHSY5Y cells and enhanced that by ACN cells. Primary tumor lesions from neuroblastoma patients tested negative for HLA-G. Neuroblastoma patients displayed a higher number of sHLA-G-secreting monocytes than healthy controls. Incubation of monocytes from normal donors with IFN-gamma or pooled neuroblastoma cell line supernatants significantly increased the proportion of sHLA-G-secreting cells. In addition, tumor cell supernatants up-regulated monocyte expression of CD68, HLA-DR, CD69, and CD71 and down-regulated IL-12 production. Our conclusions are the following: (a) sHLA-G serum levels are increased in neuroblastoma patients and correlate with relapse, (b) sHLA-G is secreted by monocytes activated by tumor cells rather than by tumor cells themselves, and (c) sHLA-G dampens anti-neuroblastoma immune responses.

in vitro neutralization of IL-10
Braun D, Galibert L, Nakajima T, Saito H, Quang VV, Rubio M, Sarfati M. (2006). "Semimature stage: a checkpoint in a dendritic cell maturation program that allows for functional reversion after signal-regulatory protein-alpha ligation and maturation signals" J Immunol 177(12):8550-9. PubMed

CD47 on live cells actively engages signal-regulatory protein-alpha (SIRP-alpha) on phagocytes and delivers a negative signal that prevents their elimination. We evaluated the biological consequences of SIRP-alpha ligation on the dendritic cell (DC) response to maturation signals and the potential interplay with the IL-10/IL-10R inhibitory pathway. At first, CD47/SIRP-alpha allowed the generation of mature migratory DCs not producing IL-12, IFN-gamma-inducible protein-10, and CCL19. Rather, they secreted neutrophils attracting chemokine CXCL5 and IL-1beta, reflecting a partial block in functional DC maturation. Afterward, semimature DCs functionally regressed in an IL-10-independent fashion toward cells that retrieved the cardinal features of immature DCs: re-expression of CCR5, loss of DC-lysosome-associated membrane protein, high endocytosis, and impaired allostimulatory functions. The global gene expression profile of IL-10 and SIRP-alpha-ligated DC demonstrated two distinct molecular pathways. IL-10R and SIRP-alpha expression were reciprocally down-regulated by CD47 and IL-10, respectively. These results emphasize that the SIRP-alpha pathway might be part of the molecular machinery used by the DC to dampen or resolve an inflammatory response in an IL-10-independent manner.

in vitro neutralization of IL-10
Kimatrai M, Blanco O, Muñoz-Fernández R, Tirado I, Martin F, Abadía-Molina AC, Olivares EG. (2005). "Contractile activity of human decidual stromal cells. II. Effect of interleukin-10" J Clin Endocrinol Metab 90(11):6126-30. PubMed

Context: Human decidual stromal cells (DSC) are myofibroblast-like cells that express alpha-smooth muscle (alpha-SM) actin, a protein associated with cell contractility. Several lines of experimental evidence in humans and mice show that antiinflammatory cytokines favor normal pregnancy, whereas Th1 and inflammatory cytokines play a role in abortion. We previously demonstrated that IL-2, a Th1 cytokine, increased the contractility of human DSC. Objective: We studied the effect of the antiinflammatory cytokines IL-10 and IL-4 on the contractility of DSC from first-trimester pregnancy. Setting and patients: We studied 10 healthy women who underwent elective vaginal termination of first-trimester pregnancy at Clínica El Sur, Málaga, and Clínica Ginegranada, Granada. Main outcome measure(s): After isolation of DSC, cell contractility was measured with the collagen gel contraction assay. alpha-SM actin was detected with Western blotting and immunofluorescence. Results: We found that IL-10, but not IL-4, increased the volume of the collagen gel matrixes in which the cytokine-treated DSC were cultured, showing that IL-10 decreased DSC contractility. By Western blotting we demonstrated that this effect was not related to an alteration in the synthesis of alpha-SM actin. Nevertheless, we observed by immunofluorescence microscopy that DSC treated with IL-10 exhibited stress fibers with a lower content of alpha-SM actin than untreated control DSC. Conclusions: IL-10 relaxes DSC by reducing the incorporation of alpha-SM actin into their stress fibers. This relaxing activity may be of relevance for the maintenance of pregnancy.

Flow Cytometry
Matsumoto K, Narita S, Rerecich T, Snider DP, O', Byrne PM. (2004). "Different profile of interleukin-10 production in circulating T cells from atopic asthmatics compared with healthy subjects" Can Respir J 11(1):33-8. PubMed

Background: Interleukin (IL)-10 is a pleiotropic cytokine released from various cells, including T cells. Although IL-10 is suggested to inhibit allergic responses, its role in asthma remains uncertain. The purpose of the present study was to compare the profile of IL-10 in circulating T cells from stable atopic asthmatics, atopic nonasthmatics and healthy controls. Methods: Peripheral blood mononuclear cells were isolated, stained with anti-CD3 and CD4/CD8 antibodies, and then processed for intracellular IL-10 detection by flow cytometry. Results: A kinetic study in healthy controls showed that stimulation with phorbol 12-myristate 13-acetate and ionomycin significantly increased the frequencies of IL-10-producing CD3+, CD4+ and CD8+ cells. Without stimulation, the frequencies of IL-10-producing CD3+, CD4+ and CD8+cells were significantly higher in asthmatics than in healthy controls, while a similar trend was observed in atopic nonasthmatics. Stimulation for 24 h significantly increased IL-10-producing CD3+, CD4+ and CD8+cells in healthy controls and atopic nonasthmatics, but not in asthmatics. Conclusions: The frequency of IL-10-producing T cells is increased in the circulation of stable atopic asthmatics compared with normal controls. The lack of enhancement in their frequency by phorbol 12-myristate 13-acetate and ionomycin in asthmatics suggests that the circulating T cells of asthmatic subjects are maximally stimulated with regards to IL-10 production; alternatively, IL-10 production by T cells from asthmatics may be regulated differently than T cells from other subjects.

Immunohistochemistry (frozen)
Melgar S, Yeung MM, Bas A, Forsberg G, Suhr O, Oberg A, Hammarstrom S, Danielsson A, Hammarstrom ML. (2003). "Over-expression of interleukin 10 in mucosal T cells of patients with active ulcerative colitis" Clin Exp Immunol 134(1):127-37. PubMed

Ulcerative colitis (UC), a chronic inflammatory bowel disease, exhibits pronounced increase of T lymphocytes in the inflamed mucosa. To understand the role of intestinal T lymphocytes in the pathogenesis of UC their cytokine production in the mucosa was analysed. Intestinal T lymphocytes of UC, Crohn's disease and control patients were analysed for cytokine mRNA levels by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) directly after isolation without in vitro stimulation. Frequencies of cytokine positive cells were determined in UC and control colon by immunomorphometry. T lymphocytes in normal colon expressed interleukin (IL)-2, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1, but not IL-4, IL-5 or IL-10. In UC, a highly significant increase in IL-10 mRNA levels in T lymphocytes and an increased frequency of IL-10 positive cells was seen in colon. IL-10 mRNA levels were also elevated in T lymphocytes of the non-inflamed ileum and correlated with disease activity at both locations. CD4+ T lymphocytes were the major source of IL-10 mRNA. IL-2, IFN-gamma and TNF-alpha mRNA levels were decreased in colonic T lymphocytes, and virtually no IL-2, IFN-gamma, TNF-alpha or TGF-beta positive cells were detected in basal lymphoid aggregates. However, scattered IL-10 positive cells were found here. Lamina propria outside the aggregates contained IL-10-, IFN-gamma, TNF-alpha and TGF-beta but not IL-2 positive cells. T cells of UC patients did not express IL-4 or IL-5. Taken, together the data suggest a generalized activation of IL-10 producing CD4+ T cells along the intestine of UC patients. The local environment seems to determine the biological consequences of elevated IL-10.

ELISA
Bacchetta R, Sartirana C, Levings MK, Bordignon C, Narula S, Roncarolo MG. (2002). "Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines" Eur J Immunol 32(8):2237-45. PubMed

Cloned T regulatory type 1 (Tr1) cells produce IL-10, TGF-beta, IFN-gamma, and very low or non-detectable levels of IL-2 and IL-4, following TCR-mediated activation. In addition, upon TCR stimulation, Tr1 cell clones up-regulate activation markers but show low proliferative responses, partially due to the suppressive effect of autocrine IL-10 and TGF-beta. Here we show that Tr1 cells have growth requirements different from those of Th1 and Th2 cells. Exogenous IL-15, and to a lesser extent IL-2, induce and support the proliferation of Tr1 cells in the absence of TCR activation. This strong cytokine response correlates with high constitutive levels of the IL-2/15Rbeta and common gamma chains expressed by Tr1 cell clones. Furthermore, suboptimal doses of IL-15, in combination with IL-2, induce a significant growth (median value: 25-fold increase in cell number) of Tr1 cell clones during a culture period of 11 days, which leads to an in vitro expansion of Tr1 cell clones comparable to that of Th1 and Th2 cell clones. Tr1 cell clones cultured in IL-15 continue to secrete immunosuppressive cytokines and to proliferate poorly upon reactivation via TCR. These findings indicate that Tr1 cells are constitutively capable of responding to cytokines and mainly to IL-15. This growth factor enables a significant in vitro expansion of Tr1 cells facilitating further biological and biochemical characterization of this unique T cell subset.

in vivo neutralization of IL-10
Feng CG, Kullberg MC, Jankovic D, Cheever AW, Caspar P, Coffman RL, Sher A. (2002). "Transgenic mice expressing human interleukin-10 in the antigen-presenting cell compartment show increased susceptibility to infection with Mycobacterium avium associated with decreased macrophage effector function and apoptosis" Infect Immun 70(12):6672-9. PubMed

Interleukin-10 (IL-10) is thought to play an important role in the regulation of microbial immunity. While T-cell-derived IL-10 has been shown to suppress cell-mediated immunity, there has been debate as to whether antigen presenting cell (APC)-derived cytokine can perform the same function in vivo. To assess the influence of APC-produced IL-10 on host resistance to mycobacterial infection, transgenic mice expressing human IL-10 under the control of the major histocompatibility complex class II promoter (hu10Tg) were infected with Mycobacterium avium, and bacterial burdens and immune responses were compared with those observed in wild-type (wt) animals. Hu10Tg mice harbored substantially higher numbers of M. avium and succumbed 16 to 18 weeks postinfection. The granulomas in infected hu10Tg mice showed marked increases in both acid-fast bacilli and host macrophages. In addition, these animals displayed a dramatic increase in hepatic fibrosis. The increased susceptibility of the hu10Tg mice to M. avium infection is independent of T-cell-produced endogenous murine IL-10, since bacterial burdens in mice derived by crossing hu10Tg mice with murine IL-10-deficient mice were not significantly different from those in hu10Tg mice. Importantly, gamma interferon (IFN-gamma) responses were not decreased in the infected transgenic animals from those in wt animals, suggesting the normal development of Th1 effector cells. In contrast, mycobacterium-induced macrophage apoptosis as well as production of TNF, nitric oxide, and IL-12p40 were strongly inhibited in hu10Tg mice. Together, these data indicate that APC-derived IL-10 can exert a major inhibitory effect on control of mycobacterial infection by a mechanism involving the suppression of macrophage effector function and apoptosis.

Functional Assays
Roth I, Fisher SJ. (1999). "IL-10 is an autocrine inhibitor of human placental cytotrophoblast MMP-9 production and invasion" Dev Biol 205(1):194-204. PubMed

During human placentation, fetal cytotrophoblast stem cells differentiate and then invade the uterine wall and its associated spiral arteries. This process anchors the placenta to the uterus and supplies maternal blood to the fetus. Cytotrophoblast invasion in vitro requires the expression of matrix metalloproteinase-9 (MMP-9). Recently, we showed that cytotrophoblasts produce interleukin-10 (IL-10), a potent immunomodulatory cytokine that could have paracrine effects on the maternal immune system. IL-10 synthesis is dramatically downregulated after the first 12 h of culture, while MMP-9 secretion is rapidly upregulated and the cells acquire an invasive phenotype. These observations prompted us to investigate whether IL-10 is an autocrine regulator of cytotrophoblast MMP-9 production. We found that the cells expressed IL-10 receptor mRNA, suggesting that autocrine effects are possible. Adding recombinant IL-10 to cytotrophoblast cultures significantly decreased the cells' MMP-9 expression at both protein and mRNA levels, but did not affect mRNA levels of the tissue inhibitor of metalloproteinase-3. Thus, IL-10 may alter the proteinase/inhibitor balance. IL-10 treatment further caused a net decrease in MMP activity, thereby reducing cytotrophoblast invasiveness. An antibody that neutralized endogenous IL-10 function had the opposite effect in all experiments. Together, these data suggest that IL-10 is an autocrine inhibitor of cytotrophoblast MMP-9 activity and invasiveness.

Functional Assays
Roilides E, Anastasiou-Katsiardani A, Dimitriadou-Georgiadou A, Kadiltsoglou I, Tsaparidou S, Panteliadis C, Walsh TJ. (1998). "Suppressive effects of interleukin-10 on human mononuclear phagocyte function against Candida albicans and Staphylococcus aureus" J Infect Dis 178(6):1734-42. PubMed

The effects of interleukin (IL)-10, a potent antiinflammatory cytokine, on human monocyte functions against two medically important pathogens, Candida albicans and Staphylococcus aureus, were studied. Incubation with 20-100 ng/mL IL-10 for 2-3 days decreased the fungicidal activity of monocytes against serum-opsonized C. albicans blastoconidia (P</=.04), reduced their capacity to damage unopsonized hyphae (P</=.006), and suppressed superoxide anion production in response to phorbol myristate acetate (P=.019) and N-FMLP (P=.04) but not to serum-opsonized blastoconidia. Paradoxically, IL-10 enhanced phagocytic activity of monocytes against serum-opsonized blastoconidia (P<.01). In addition, IL-10-treated monocytes demonstrated decreased bactericidal activity (P=.046) but no change in bacterial phagocytosis. These findings demonstrate an overall suppressive role of IL-10 on human monocyte function against C. albicans and S. aureus and may have important implications in the use of this cytokine.

Flow Cytometry, Functional Assays
Bellinghausen I, Metz G, Enk AH, Christmann S, Knop J, Saloga J. (1997). "Insect venom immunotherapy induces interleukin-10 production and a Th2-to-Th1 shift, and changes surface marker expression in venom-allergic subjects" Eur J Immunol 27(5):1131-9. PubMed

The current study was carried out to elucidate the immunoregulatory changes induced by venom immunotherapy (VIT) in bee or wasp allergic subjects. All subjects included in this study had a history of severe systemic allergic reactions to stings of the respective insect as well as positive skin tests with the respective venom or venom-specific IgE in the sera. Parameters assessed in peripheral blood mononuclear cells (PBMC) before and after initiation of VIT (rush therapy reaching a maintenance dose of 100 micrograms venom injected subcutaneously within 1 week) were expression of CD3, CD4, CD8, CD45RA, CD45RO, interleukin (IL)-2 receptor (R) alpha, IL-4R, IL-12R, Fc epsilon RII, CD40, and CD40 ligand (CD40L), cells producing interferon (IFN)-gamma and IL-10 after stimulation with phorbol 12-myristate 13-acetate + ionomycin in the presence of monensin measured by flow cytometry; secretion of IFN-gamma, IL-4, and IL-10 measured by ELISA (IFN-gamma and IL-10 were additionally measured by PCR), and proliferation after stimulation with the respective venom. Significant decreases were observed after VIT for proliferative response to venom and venom + IL-4, IL-4 secretion, Fc epsilon RII, CD40, and CD40L expression. Significant increases were observed after VIT for IFN-gamma concerning the amount secreted and the number of producing cells, and IL-10, IL-10 was mainly produced by CD4+ cells that were negative for IFN-gamma, but some double-positive (IL-10 and IFN-gamma) cells were always detected. Addition of blocking anti-IL-10 antibodies, but not isotype control antibodies, prevented down-regulation of proliferation (but not IL-4 secretion) and further enhanced IFN-gamma secretion after VIT. These data indicate that in insect venom allergic subjects, VIT not only induces a rapid shift in cytokine expression from Th2 to Th1 cytokines, but also leads to induction of the immunosuppressive cytokine IL-10, which may be important for the limitation of potentially harmful allergen-specific Th1 responses. The described changes in cytokine expression may be responsible for subsequent increases in allergen-specific IgG and decreases in IgE production, as well as suppressive activity observed in earlier studies.

Immunohistochemistry (frozen)
Buhl L, Søgaard H. (1997). "Immunohistochemical expression of IL-10 in mycosis fungoides" Exp Dermatol 6(4):195-8. PubMed

Immunoreactivity of the cytokine IL-10 has been investigated in situ in mycosis fungoides (MF). Expression of IL-10 was detected using immunohistochemistry in skin biopsies (n = 8) and T-cell lines (n = 2) from mycosis fungoides patients. IL-10 positivity was seen in the dermal cell infiltrates and in T-cell lines in mycosis fungoides. The dermal IL-10 reaction in a skin biopsy from an active lesion in MF indicates the possibility for disease progression.

ELISA
Secrist H, Levy S, DeKruyff RH, Umetsu DT. (1996). "Ligation of TAPA-1 (CD81) or major histocompatibility complex class II in co-cultures of human B and T lymphocytes enhances interleukin-4 synthesis by antigen-specific CD4+ T cells" Eur J Immunol 26(7):1435-42. PubMed

We have previously shown that CD4+ T cells from allergic individuals are predisposed to producing interleukin (IL)-4 in response to allergens. IL-4 production could be modulated by antigen concentration as well as by the type of antigen-presenting cells (APC), with B lymphocytes inducing greater quantities of IL-4 than monocytes. Using this system we examined IL-4 synthesis after culture of CD4+ T cells with B cells, monocytes, or both, as APC in the presence of allergen and a monoclonal antibody against CD81 (TAPA-1), a member of the TM4 superfamily of proteins that regulates activation, proliferation and trafficking of B cells. Addition of anti-CD81 mAb during culture enhanced IL-4 synthesis by 2- to 70-fold over that using an isotype-matched control mAb. Furthermore, anti-CD81 mAb enhanced IL-4 synthesis in CD4+ T cells only when CD4+ T cells were cultured with B cells but not monocytes as APC, indicating that anti-CD81 mAb affected IL-4 synthesis in T cells via interactions with B cells. However, pretreatment of either population separately with anti-CD81 mAb prior to culture had no effect on subsequent IL-4 synthesis, suggesting a requirement for temporal or cooperative interactions between T and B lymphocytes. In addition, anti-CD81 mAb enhanced IL-4 production but reduced CD4+ T cell antigen-specific proliferation, demonstrating that IL-4 production and proliferation by CD4+ T cells were inversely related. Finally, mAb to major histocompatibility complex class II but not to anti-CD19 also enhanced IL-4 synthesis when B lymphocytes were used as APC. In all instances, enhancement of CD4+ IL-4 synthesis correlated with the presence of large cell aggregates in T-B lymphocyte cocultures. These results indicate that the capacity of B cells to induce IL-4 can be significantly enhanced by ligation of particular molecules on their surface and should aid in the design of treatments for diseases in which modulation of the cytokine profile would be beneficial.

in vitro neutralization of IL-10
Williams K, Dooley N, Ulvestad E, Becher B, Antel JP. (1996). "IL-10 production by adult human derived microglial cells" Neurochem Int 29(1):55-64. PubMed

Microglia, a population of central nervous system (CNS) macrophages, have been demonstrated to support immune accessory and effector functions in the CNS. Numerous studies support the role of microglia in CNS development and pathology, where activation of microglia is consistently noted. The current study investigated microglial immune functions under basal and activation conditions and assessed the ability of interleukin-10 (IL-10), added exogenously or produced by microglia, to down-regulate microglial functions. This report demonstrates that microglia from the adult human brain produce IL-10 following interferon-gamma/lipopolysaccharide activation. Functionally, recombinant human IL-10 down-regulated basal HLA-DR expression by microglia and inhibited, in a dose-dependent response, the ability of microglia to stimulate CD4+ T-cells in antigen presentation assays. These data, together with recent observations of the inhibition of experimental allergic encephalomyelitis (EAE) following IL-10 administration and reduced CNS infection by Listeria monocytogenes after anti-IL-10 treatment, suggest that IL-10 production by microglia may have important immune-regulatory functions in CNS disease and disease models.

in vitro neutralization of IL-10
Armstrong L, Jordan N, Millar A. (1996). "Interleukin 10 (IL-10) regulation of tumour necrosis factor alpha (TNF-alpha) from human alveolar macrophages and peripheral blood monocytes" Thorax 51(2):143-9. PubMed

Background: Regulation of the inflammatory response within the human lung is essential to prevent this important part of the normal host defence response becoming a pathological process. Tumour necrosis factor alpha (TNF-alpha) is a cytokine involved in the pathogenesis of shock and in granuloma formation, tissue necrosis, and fibrosis in many organ systems including the lung. Interleukin 10 (IL-10) has been proposed as having an inhibitory effect on the production of several inflammatory cytokines including TNF-alpha. Methods: The effect of IL-10 administration on TNF-alpha production was explored in human alveolar macrophages and peripheral blood monocytes from matched individuals. The effects of IL-10 on TNF-alpha protein production were determined by sandwich enzyme linked immunosorbant assay (ELISA), whereas the TNF-alpha mRNA response was established by Northeren blotting using a TNF-alpha specific oligonucleotide probe. The protein synthesis inhibitors actinomycin D and cyclohexamide were utilised to monitor IL-10 effects on mRNA degradation and de novo protein synthesis, respectively. Results: The lipopolysaccharide-mediated TNF-alpha production in alveolar macrophages was reduced from 3.508 (0.629) to 2.035 (0.385) ng/ml by 100 U/ml IL-10. Lipopolysaccharide-induced TNF-alpha production in peripheral blood monocytes was reduced from 2.035 (0.284) to 0.698 (0.167) ng/ml. TNF-alpha gene expression was also inhibited in both alveolar macrophages and peripheral blood monocytes; lipopolysaccharide-induced TNF-alpha mRNA was reduced by 47.8 (15.2)% and 83.1 (4.2)%, respectively, by IL-10. The IL-10 mediated suppression of TNF-alpha mRNA was unaffected by addition of cyclohexamide, suggesting that de novo protein synthesis was not required for TNF-alpha inhibition. mRNA stability experiments indicated no acceleration in lipopolysaccharide-induced TNF-alpha mRNA degradation in response to IL-10. Conclusions: These findings suggest that IL-10 is a potent inhibitor of TNF-alpha expression and release from alveolar macrophages and peripheral blood monocytes, and thus it may have an important role in the cytokine network of the pulmonary immune response.

Functional Assays
Clerici M, Wynn TA, Berzofsky JA, Blatt SP, Hendrix CW, Sher A, Coffman RL, Shearer GM. (1994). "Role of interleukin-10 in T helper cell dysfunction in asymptomatic individuals infected with the human immunodeficiency virus" J Clin Invest 93(2):768-75. PubMed

The loss of T helper cell (TH) function in asymptomatic HIV type 1-infected individuals occurs before the decline in CD4+ T cells. At least part of the loss in TH function results from changes in immunoregulatory cytokine profiles. To investigate the role of IL-10 in such dysregulation, we tested whether: (a) expression of IL-10-specific mRNA would be upregulated in PBMC from asymptomatic, HIV-infected (HIV+) individuals; (b) PBMC from these same individuals would produce increased levels of IL-10 when stimulated in vitro with phytohemagglutinin; and (c) defective antigen-specific TH function could be restored by anti-IL-10 antibody. We observed that IL-10-specific mRNA was marginally upregulated, and increased levels of IL-10 were produced by PBMC from HIV+ individuals compared with PBMC from uninfected individuals. Those individuals whose TH function was more severely compromised produced higher levels of IL-10. Additionally, defective antigen-specific TH function in vitro could be reversed by anti-IL-10 antibody, including the response to HIV envelope synthetic peptides. Furthermore, the antigen-specific TH responses of HIV-uninfected PBMC could be reduced with IL-10, a process reversed by anti-IL-10. These results confirm that the early loss of TH function in HIV+ individuals is due at least in part to cytokine-induced immune dysregulation, and support the hypothesis of a switch from a predominant type 1 state to a predominant type 2 condition in HIV infection.

in vitro neutralization of IL-10
Krüger-Krasagakes S, Krasagakis K, Garbe C, Schmitt E, Hüls C, Blankenstein T, Diamantstein T. (1994). "Expression of interleukin 10 in human melanoma" Br J Cancer 70(6):1182-5. PubMed

The expression of interleukin 10 (IL-10) mRNA in human malignant melanoma was investigated by reverse transcriptase polymerase chain reaction analysis. Selective expression of IL-10 mRNA in tissues of primary melanomas and melanoma metastases was found in comparison with normal skin. In addition, strong expression of IL-10 mRNA and of biologically active IL-10 was detected in 3 out of 13 melanoma cell lines. Normal melanocytes consistently expressed low levels of IL-10 mRNA but did not produce detectable IL-10 protein, nor did keratinocytes or fibroblasts. The production of biologically active IL-10 by melanoma cell lines suggests that IL-10 mRNA in melanoma lesions may derive at least in part from the tumour cells themselves. Tumour-infiltrating cells, however, could also be a source of IL-10 in melanoma tissues. The presence of IL-10 in melanoma lesions may contribute to the postulated 'paralysis' of an anti-melanoma immune response.

Immunohistochemistry (frozen), in vitro neutralization of IL-10
Andersson J, Abrams J, Björk L, Funa K, Litton M, Agren K, Andersson U. (1994). "Concomitant in vivo production of 19 different cytokines in human tonsils" Immunology 83(1):16-24. PubMed

Accumulating data indicate that cytokines, peptides involved in regulation of both physiological and pathological immune responses, are produced predominantly at the site of local antigen stimulation. Cytokine-producing cells were detected at the protein level in human tonsil tissue obtained from children with recurrent tonsillitis or infectious mononucleosis (IM). Concomitant production of 19 different human cytokines, interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1 receptor antagonist (ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, tumour necrosis factor-alpha (TNF-alpha), TNF-beta, interferon-gamma (IFN-gamma) and transforming growth factor-beta 1-3 (TGF-beta 1-3), was identified at a single-cell level by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific antibodies (Ab). Fresh frozen sections were fixed with 4% paraformaldehyde and permeabilized by 0.1% saponin treatment, eluting cholesterol from the cell-surface membrane and the Golgi complex. The intracellular localization of all cytokines, except IL-1 and IL-1ra, was demonstrated by a characteristic local cytoplasmic perinuclear configuration in producer cells. In addition, the immunoreactivity for certain cytokines (IL-2, IL-4, IL-5, G-CSF and GM-CSF) was expressed on the cell membranes and extended over a large extracellular area encompassing the producer cell. Localization of the cytokine to the Golgi organelle was established by co-staining with a monoclonal antibody (mAb) specific to the Golgi complex. Both the extra- and intracellular cytokine staining reactions could be blocked by preincubation of the cytokine-specific Ab with the corresponding purified natural or recombinant cytokine. A complex cytokine pattern was established in both groups studied, where most T-helper type 1 (Th1) and Th2 lymphokines were expressed in the tonsils but at different frequencies and localizations. Cells expressing IL-4, IL-5, IL-10 and IL-13, (Th2 response) were evident at higher frequencies in recurrent tonsillitis compared to sections from IM, which were associated with a more pronounced IL-2, IFN-gamma and TNF-beta expression.

in vitro neutralization of IL-10
Ghalib HW, Piuvezam MR, Skeiky YA, Siddig M, Hashim FA, el-Hassan AM, Russo DM, Reed SG. (1993). "Interleukin 10 production correlates with pathology in human Leishmania donovani infections" J Clin Invest 92(1):324-9. PubMed

We have found that an important Th2 cytokine, IL-10, is produced by tissues from patients acutely infected with Leishmania donovani. In all individuals tested, IL-10 mRNA production was increased in lymph nodes taken during acute disease over that observed in postacute samples. In contrast, both pre- and posttreatment lymph nodes had readily detected mRNA for IFN-gamma and IL-2. A down-regulating effect of IL-10 on leishmania-induced proliferative responses was demonstrated when Hu rIL-10 was added to cultures of PBMC from clinically cured individuals. PBMC from individuals with acute visceral leishmaniasis responded to stimulation with leishmania lysate by producing IL-10 mRNA. Simultaneously cultured PBMC collected from the same patients after successful chemotherapy produced no detectable IL-10 mRNA after leishmania antigen stimulation. Neutralizing anti-IL-10 mAb added to PBMC from patients with acute visceral leishmaniasis markedly increased the proliferative response to leishmania lysate. Finally, we observed mRNA for IL-10 and IFN-gamma concurrently in a lesion from a patient with post-kala-azar dermal leishmaniasis (PKDL). These results indicate the production of IL-10 during L. donovani infection, and suggest a role for this cytokine in the regulation of immune responsiveness during visceral leishmaniasis.

in vitro neutralization of IL-10
de Waal Malefyt R, Haanen J, Spits H, Roncarolo MG, te Velde A, Figdor C, Johnson K, Kastelein R, Yssel H, de Vries JE. (1991). "Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression" J Exp Med 174(4):915-24. PubMed

Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma- or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Furthermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.

in vitro neutralization of IL-10
de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE. (1991). "Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes" J Exp Med 174(5):1209-20. PubMed

In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF alpha), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon gamma (IFN-gamma), LPS, or combinations of LPS and IFN-gamma at the onset of the cultures, strongly inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNF alpha and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.