Effects of single intravenous doses of recombinant human interleukin-10 on subsets of circulating leukocytes in humans.

Recombinant human interleukin-10 (rhIL-10) is a potent and specific immunomodulatory agent which inhibits endotoxin-stimulated pro-inflammatory cytokine production by monocytes, blocks T-lymphocyte activation by antigen presenting cells, and modulates T(H)1/T(H)2 balance in immune responses. In previous clinical trials, rhIL-10 administered to healthy volunteers induced rapid and transient elevations of neutrophil and monocyte counts and reductions of lymphocyte counts in addition to suppression of endotoxin-stimulated whole blood cytokine synthesis. We sought to better characterize the effects of rhIL-10 on immunophenotypically defined subsets of circulating leukocytes that could be relevant to its immunomodulatory effects. Healthy volunteers were given single doses of 10 microg/kg rhIL-10 (n = 8) or equivalent placebo (n = 4) by intravenous injection. Significant changes of circulating leukocytes included transiently increased neutrophils and monocytes with parallel increases of CD33+ and CD14+ cells. Total lymphocytes as well as total CD3+, CD3+/CD4+ and CD3+/CD8+ cells transiently decreased. Mean fluorescence intensity of CD11a (integrin alpha-chain subunit of lymphocyte function antigen-1, LFA-1) on lymphocytes transiently but significantly decreased, suggesting a mechanism for transient alteration of lymphocyte trafficking. In addition, mean fluorescence intensity of HLA-DR (major histocompatibility class II) on CD14+ cells (predominantly monocytes) transiently but significantly decreased, implying a possible alteration of antigen presenting function. Further study will be required to elucidate the immunomodulatory roles and potential clinical significance of these hematologic changes in therapeutic trials of rhIL-10 in patients with chronic inflammatory and autoimmune diseases.

Interleukin-10 attenuates experimental fetal growth restriction and demise.

Premature labor, fetal demise, and fetal growth restriction are accompanied by indices of inflammation or infection of the uteroplacental unit. To understand whether these events are causally related, we established an animal model of fetal demise and growth restriction and evaluated the potential utility of the anti-inflammatory cytokine interleukin-10 (IL-10). We administered low-dose endotoxin (lipopolysaccharide, or LPS, 100 microg/kg, i.p.) to third trimester rats (gestational days 14-20). Control rats received normal saline. A third group received IL-10 (100 microg/kg; s.c.) concomitantly with LPS for 7 prenatal days. Cytokine gene expression (IL-10 and TNF-alpha) was evaluated by RT-PCR and tissue levels (TNF-alpha) were determined by ELISA. Apoptosis was evaluated by TdT-mediated dUTP nick end labeling immunohistochemistry, and nitric oxide (NO) levels were quantified by microelectrode electrochemical detection in explants in culture media. LPS exposure resulted in 43% fetal demise and reduced the size of the surviving fetuses. Placental weight was not altered by LPS. IL-10 attenuated the LPS-induced fetal death rate (to 22%) and growth restriction (P<0.05). In normal rats, IL-10 did not affect fetus size or the incidence of resorptions, although placental size was marginally smaller. Increased uterine TNF-alpha content and NO release and apoptosis of uterine epithelia and muscularis were hallmarks of the LPS model. All were normalized by IL-10. IL-10 may represent a new therapeutic option for the treatment of a variety of perinatal complications. Benefit may result from the suppression of TNF-alpha- and NO-mediated cell death.

The inhibitory effects of topically active glucocorticoids on IL-4, IL-5, and interferon-gamma production by cultured primary CD4+ T cells.

This study was conducted to directly compare the in vitro efficacy and potency of several glucocorticoids in inhibiting T-cell cytokine production. The glucocorticoids tested were fluticasone propionate, budesonide, triamcinolone acetonide, and beclomethasone dipropionate, which are currently inhaled therapies for the treatment of allergic airway disease. Also used were betamethasone phosphate and the newly developed mometasone furorate. With a novel cell culture system, purified peripheral blood CD4+ T cells from normal donors were stimulated with immobilized anti-CD3 and soluble anti-CD28 monoclonal antibodies to induce high levels of IL-4, IL-5, and interferon-gamma. By cell sorting, it was found that the IL-5 produced originated from memory cells, whereas both memory and naive cells produced interferon-gamma. Mometasone and fluticasone inhibited IL-5 and IL-4 similarly (mometasone IL-5 inhibitory concentration of 50% = 0.27 +/- 0.1 nmol/L and IL-4 = 0.19 +/- 0.08 nmol/L). For both cytokines, the results indicate that mometasone and fluticasone were more potent than beclomethasone, triamcinolone, budesonide, and betamethasone. Of clinical importance is the finding that all steroids demonstrated less efficacy versus interferon-gamma than IL-4 and IL-5. Glucocorticoid reduction of Th2 cytokines with lesser effects on interferon-gamma would serve to reverse the exaggerated Th2 response that contributes to pathophysiology observed in allergic disease. Therefore the use of topically active glucocorticoids with low systemic bioactivity for the treatment of allergic inflammation may be particularly effective in modulating the cytokine activity that is an important component of the allergic response.

Anti-inflammatory properties of interleukin-10 administration in hapten-induced colitis.

Therapeutic efficacy of interleukin-10 administration in colonic inflammation was assessed in rats. Following intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS), subcutaneous administration of 1-1000 micrograms/kg per day interleukin-10, or a placebo (0.9% NaCl) was commenced and continued for 5 days. Interleukin-10 administered at 1, 10 and 100 micrograms/kg per day significantly reduced myeloperoxidase activity by 34, 57, and 28%, respectively, compared to the placebo-treated group, which was paralleled by an attenuation of colonic tumor necrosis factor alpha (TNF-alpha) content. In contrast, the severity of mucosal necrosis was not affected by interleukin-10 administration at the dose range used. In addition, the 10-fold elevation in nitric oxide release, 5-fold rise in colonic nitrite production and enhanced expression of inducible nitric oxide synthase, associated with TNBS colitis, was not suppressed by interleukin-10. Interleukin-10 gene expression was elevated during the first 14 days of TNBS colitis. We conclude that 5 days administration of interleukin-10 in TNBS colitis displays mild anti-inflammatory properties which were not mediated via a nitric oxide-dependent pathway, but may involve TNF-alpha.

Detection of cell-surface antigens using antibody-conjugated fluorospheres (ACF): application for six-color immunofluorescence.

Antibody-conjugated fluorospheres (ACF) were used to phenotype murine leukocytes by flow cytometric analysis. Multicolor immunofluoresence (beyond simultaneous 4-color analysis) is limited by the availability of specific antibody-fluorochrome chrome conjugates and even further restricted by the spectral emission overlap of many of the fluorochromes when used in combination. Fluorospheres possessing unique excitation/emission spectra can provide much needed versatility to existing protocols of multicolor fluorescence. SKY BLUE (647 nm excitation, 730 nm emission) fluorospheres conjugated to CD11b monoclonal antibody were used in combination with the monoclonal antibodies IAd-FITC, L3T4 (CD4)-PE, LYT2 (CD8)-APC, THY1.2 (CD90)-biotin and B220 (CD45R)-RED613 for the simultaneous detection of six distinct cell-surface antigens in a mixed cell population. All fluorescence signals were resolved, and comparison of results from five-, six- and single-color samples indicated that the percentages of cells positive for specific surface antigens were equivalent.

Allo-7: a new fluorescent tandem dye for use in flow cytometry.

This report describes the development of a novel tandem dye by combining allophycocyanine (APC) and cyanine dye indotricarbocyanine (CY7) to create ALLO-7 for use in flow cytometry. The APC donor fluorophore was excited at 647 nm and, through resonance energy transfer to the CY7 acceptor, produced fluorescence at > 780 nm. To test the applicability of this tandem in single and multicolor immunofluorescence, a streptavidin conjugate of the tandem (SA-ALLO-7) was used for the detection of cell surface antigens on human peripheral blood leukocytes (PBL) by indirect immunofluorescence. Human PBL were stained with CD4/ GaM-APC, CD3-fluorescein isothiocyanate (FITC), CD14-phycoerythrin (PE), CD19-energy-coupled dye (phycoerythrin-Texas Red) (ECD), and CD8-biotin with SA-ALLO-7 and analyzed for fluorescence on a FACS Vantage using dual-laser excitation (488 and 647 nm). The results indicated that the percentage of cells positive for each of the surface antigens was comparable for single-color controls and multicolor samples. The ALLO-7 fluorescence, which was collected with a 730-shortpass dichroic mirror and a 790/50-bandpass filter, was clearly resolved from the APC fluorescence and that from FITC, PE, and ECD. The SA-ALLO-7 exhibited minimal nonspecific binding to PBL monocytes. However, the specific binding of the tandem to high-density antigens was clearly identified by positive fluorescence. This unique tandem reagent, ALLO-7, provided the capability for dual-color immunofluorescence with a 647-nm laser line (or a helium neon laser at 633 nm) and provides the potential to perform three-color analysis with a dye-head laser (Texas Red, APC, ALLO-7).

T cells are necessary for Th2 cytokine production and eosinophil accumulation in airways of antigen-challenged allergic mice.

In a murine model of pulmonary inflammation, aerosolized antigen challenge of sensitized B6D2F1 mice leads to eosinophil accumulation within the lungs. Little is known of the role of T cells and their cytokine products in these allergic animals. In this study, we show that T cells migrate into the lungs in response to antigen challenge and are necessary for local production of cytokines (IL-4 and IL-5) important in B and T cell development as well as eosinophil activation and differentiation. Flow cytometry revealed an increase in the percentage of Thy1+ T cells but not in B220+ B cells in bronchoalveolar lavage fluid after challenge when compared to unchallenged mice. Although there was an increase in both T cell subsets, there were twice as many CD4+ cells as CD8+ cells at 24 hr and after 48 hr the CD4+ subset predominated. The CD4+ T lymphocytes were CD44+ CD45RBlo indicating an activated/memory phenotype and tracheobroncheal lymph node cells obtained from challenged mice proliferated in a dose-dependent manner in response to antigen stimulation in vitro. Reverse transcriptase-polymerase chain reaction analysis of lung tissue-derived RNA indicated an increase in Th2-like cytokines. IL-4 and IL-5 steady-state mRNAs were at peak levels 6 hr after challenge, while no consistent increase was found for IFN-gamma mRNA levels. Treatment with the glucocorticoid betamethasone just prior to challenge reduced the levels of cytokine mRNA as well as the eosinophil influx. In vivo depletion of T cells from sensitized mice reduced pulmonary eosinophilia as well as the expression of IL-4, IL-5, and IFN-gamma steady-state mRNAs in the lungs of sensitized and challenged mice. These results indicate that T cells migrating into the lungs of mice after antigen challenge play an important role in the production of Th2-like cytokines and the accumulation of eosinophils in bronchial fluids.

Inhibitory effects of recombinant human interleukin 10 on disease manifestations in a P-->F1 model of acute graft versus host disease.

Interleukin 10 (IL-10) is a cytokine with both antiinflammatory and immunosuppressive properties. In the present study, we have examined the effects of recombinant human IL-10 (rHuIL-10) on the development of acute graft-vs.-host disease (GVHD) in unirradiated (C57B1/6JxA/J) F1 recipients of parental A/J lymphocytes. rHuIL-10 (2.5 to 100 micrograms/mouse administered subcutaneously) caused a significant reduction in splenomegaly in GVH mice. GVH splenocytes exhibited an augmented capacity to produce IFN-gamma when stimulated in culture with Con A or LPS. The IFN gamma produced in response to LPS stimulation was found to be derived from CD4+ and CD8+ T cells with little or no contribution from the NK1.1+ subpopulation of the GVH spleen. Treatment with IL-10 in vivo was found to diminish the capacity of splenocytes to produce IFN gamma when stimulated with LPS but not with Con A. IL-10 did not protect GVH mice from a lethal dose of LPS but caused a marked reduction in the serum TNF alpha response triggered by the LPS challenge. We conclude that IL-10 may be useful in controlling those clinical manifestations of acute GVHD that arise as a result of the activities of proinflammatory cytokines such as IFN gamma and TNF alpha.

Recombinant human IL-10 prevents the onset of diabetes in the nonobese diabetic mouse.

The role of IL-10 in the pathogenesis of autoimmune diabetes mellitus was assessed in the nonobese diabetic (NOD) mouse. In these studies the effect of IL-10 was determined on three parameters of diabetes: The development of hyperglycemia, the development of insulitis, and the production of insulin by beta cells. Initial experiments investigated the effect of anticytokine antibodies on the development of disease. These results indicated that monoclonal anti-IFN-gamma antibody greatly reduced the incidence of hyperglycemia in female NOD mice, while anti-IL-4, IL-5, and IL-10 were ineffective. In subsequent studies, daily subcutaneous administration of IL-10, a known potent inhibitor of IFN-gamma production by TH1 T cells, to 9 and 10-week-old NODs was shown to delay the onset of disease and significantly reduce the incidence of diabetes. Histopathology performed on pancreatic tissue demonstrated that treatment with IL-10 reduced the severity of insulitis, prevented cellular infiltration of islet cells, and promoted normal insulin production by beta cells. Taken together these results indicate IL-10 suppresses the induction and progression of autoimmune pathogenesis associated with diabetes mellitus and suggest a potential therapeutic role for this cytokine in this autoimmune disease.

Simultaneous measurement of five-cell surface antigens by five-colour immunofluorescence.

Multicolour immunofluorescence and flow cytometry were used for simultaneous measurement of five-cell surface antigens on murine spleen cells. We have been able to quantitate T-cells, T-cell subsets, B cells, and expression of the activation marker I-Ad from a single sample using four directly conjugated monoclonal antibodies LYT2-APC, L3T4-PE, B220-RED613, I-Ad-FITC and one indirect step THY1.2-biotin/streptavidin-Cascade Blue. Three excitation wavelengths were used (488 nm, 647 nm, and U.V. 351-364 nm) for fluorescence measurements. The combination of fluorochromes used provided good resolution such that all five fluorescence signals were spectrally resolved. The percentage of cells positive for expression of a specific cell surface marker were almost identical for single-colour samples and the five-colour analysis, differing by only 0.3-1.5 percentage points.

Tracking of murine spleen cells in vivo: detection of PKH26-labeled cells in the pancreas of non-obese diabetic (NOD) mice.

Flow cytometry was used to track the in vivo migration of PKH26-labeled donor spleen cells from diabetic NOD mice that were injected into non-diabetic recipient NOD mice. Flow cytometric analysis of recipient mouse tissues revealed that the donor cells were present in the peripheral blood, spleen and lymph nodes 24 h following injection and could still be detected after 28 days. PKH26(+) cells were also detectable in the pancreas 7 days after injection. Phenotypic analysis of the PKH26(+) cells that migrated into these target organs and tissues showed that the major cell population detected was Thy1.2(+) T-lymphocytes, predominantly the Thy1.2(+)/L3T4(+) subpopulation, but Thy1.2(+)/Lyt2(+) cells as well as B220(+) cells (B lymphocytes) were also present.

Cytokine gene expression in mice undergoing chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) can be induced in B6D2F1 mice by injection of parental DBA/2 lymphoid cells. Stimulation of donor T cells by host MHC antigens leads to the stimulation of host B cells. Little is known of the lymphokines produced during such a reaction. This study was designed to directly measure the levels of mRNA for interferon-gamma (IFN-gamma), interleukin 2 (IL-2), IL-4, IL-5, and IL-10, as well as several other genes, using semiquantitative polymerase chain reaction (PCR). Semiquantitative PCR was reproducible and signals generated were dependent on the amount of specific RNA or cDNA in each reaction. Early during the progression of GVHD (2 days after the first injection of parental cells) there was little increase in IL-10 mRNA, a slight increase in IL-4 mRNA, and a dramatic increase in IL-2 mRNA. In addition, IL-2 bioactivity was demonstrated in supernatants from GVH splenocytes cultured in vitro for 24 h. Later in the response (1 week after the second and final injection of parental cells) IL-4 mRNA levels were elevated as they were earlier while IL-10 mRNA levels were dramatically increased. IL-2 mRNA levels were no different in mice undergoing GVHD than in normal mice at this time. IFN-gamma mRNA was detectable both early and late, although at similar levels in normal mice and mice undergoing GVHD. At both times examined, IL-4 was below the limits of detection by bioassay and IFN-gamma, IL-4, IL-5 and IL-10 were below the limits of detection by ELISA. Further studies showed that a majority of the IL-4 and IL-10 mRNA found elevated in GVH mice were produced by Thy1.2+ T cells, with small amounts from B220+ B cells. In addition, the detectable IFN-gamma mRNA found in GVH mice at this later time also was produced by Thy1.2+ T cells, with small amounts from B220+ B cells.

Detection of in vivo-induced IL-1 mRNA in murine cells by flow cytometry (FC) and fluorescent in situ hybridization (FISH).

Flow cytometry (FC) and fluorescent in situ hybridization (FISH) were used to detect in vivo induced IL-1 alpha (a) messenger RNA. Spleen cells and thioglycollate-induced peritoneal exudate cells (PEC) were harvested from Balb/C mice three hours after intraperitoneal injection of 20 micrograms LPS. Cells from each population were phenotyped for MAC or IAd, fixed in 4% paraformaldehyde and then permeabilized with 70% ETOH. RNA-RNA FISH was performed by incubating suspended cells at 37 degrees C for 17 hours with biotinylated sense IL-1a, antisense IL-1a or antisense IL-2 probes in 50% formamide. Hybridized cells were washed in 2X SSC, treated with RNAse, stained with avidin conjugated to fluorescein (FITC) or allophycocyanin (APC) and analyzed immediately by FC. Initially, avidin-FITC was used to detect hybridized probe. Dual fluorescent FC analysis of IL-1a mRNA expression in LPS stimulated IAd+ cells showed an increase in mean fluorescent intensity (MFI) of 51 log channels (0.25 logs) when compared to unstimulated cells. Additionally, induction of specific IL-1a mRNA expression in cells from LPS treated animals was illustrated by increases in percent positive cells (24%) and in equivalent soluble fluorescein molecules (ESFM) bound (47%) when compared to cells from vehicle treated mice. Unhybridized cells and cells hybridized with control antisense IL-2 probe did not exhibit increases in MFI or ESFM. In subsequent experiments on MAC+ PEC, the use of avidin-APC to detect bound probe resulted in a greater separation between the FISH signals of antisense IL-1a and control sense probe (121 log channels, 0.6 logs) than that seen with FITC.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of recombinant murine GM-CSF (rmuGM-CSF) induced changes in the distribution of specific cell populations in vivo.

The changes in the distribution of granulocytes, monocytes, and lymphocytes in various tissue compartments following subcutaneous (SC) administration of recombinant murine GM-CSF (rmuGM-CSF) in vivo was determined by flow cytometry in time course studies. Balb/c mice were given single, daily SC injections of 1 or 4 micrograms of rmuGM-CSF for 10 days. Flow cytometric analysis was performed on bone marrow (BMC), peritoneal exudate (PEC), and peripheral blood (PBC) cell preparations from mice treated for 1, 3, and 10 days. Dual fluorescence was employed to gate on leukocytes (T200+) and analyze for Ig+, Thy 1.2+, MAC+, and 8C5+ (granulocytes) cells. The analyses indicated that SC-rmuGM-CSF increased the percentage of 8C5+ cells in PBC after 1 day of treatment. However, significant changes in the cell composition of PEC and BMC were not observed until day 10 of treatment and included increases in 8C5+ cells and the myeloid cell population, respectively. Side scatter analysis (cell density) of PBC and PEC indicated that the percentage of the granulocytic cell population increased significantly in rmuGM-CSF treated mice. The changes observed in PEC and BMC appeared to be dose-related whereas those observed in PBC were not. These data clearly demonstrate the utility of flow cytometric analyses for detecting selective effects of cytokines on cell populations that are involved in host defense mechanisms.

Inhibition of collagen II-induced arthritis in mice--a comparison of the effects of Sch 24937, immunosuppressants and nonsteroidal antiinflammatory drugs on the clinical expression of disease.

Previous studies from this laboratory have described Sch 24937 as a potent immunosuppressive agent that is particularly effective in suppressing humoral immune responses in mice. These findings prompted an evaluation of the effects of Sch 24937 in type II collagen-induced arthritis in mice where disease manifestations include the development of a strong humoral response to the collagen antigen. Sch 24937 reduced the incidence and severity of arthritis in collagen sensitized mice which appeared to be directly related to the immunosuppressive properties of the drug. However in contrast to the steroid betamethasone which also exhibited immunosuppressive activity, Sch 24937 did not prevent the changes occurring in the lymphocyte population of the draining lymph nodes of mice immunized with type II collagen. While the exact mechanism of the immunosuppressive activity of Sch 24937 remains to be elucidated, its mode of action in suppressing arthritis differs at least to some extent from that of a steroid.

Inhibition of graft-vs-host induced immunodeficiency with immunosuppressive therapy.

The pathologic features of the acute graft-vs-host disease occurring in unirradiated (C57Bl/6 X A/J)F1 mice injected intravenously with lymphocytes from the C57Bl/6 parent are similar to those reported for other parental----F1 hybrid combinations. When stimulated in culture with concanavalin A, lipopolysaccharide or alloantigen, spleen cells from B6AF1 mice that had been injected 11 days previously with B6 lymphocytes exhibited proliferative responses that were drastically reduced in comparison to the responses of spleen cells from F1 hosts injected with syngeneic lymphocytes. IL2 production in GVH spleen cell cultures was also diminished. Proliferative responses and IL2 production were partially restored in mice given immunosuppressive therapy with azathioprine, cyclosporin A or Sch 24937 a drug whose inhibitory effects on cellular and humoral immune responses in mice have recently been described. Phenotypic analyses by flow cytometry of the GVH splenocyte population indicated that the most consistent change in the GVH spleen was the appearance of an Lyt2+ L3T4+ T cell subset which in the majority of experiments was accompanied by an increase in cells expressing only the Lyt2 antigen. Both subpopulations were reduced in mice that had recovered immunological responsiveness following immunosuppressive therapy. The results suggest that in this GVH model the development of an immunodeficient state is directly related to the induction of an active T suppressor cell population and that such cells are effectively eliminated from the splenocyte population following treatment with some immunosuppressive drugs.