This unit describes a general method for immunoenzymetric assay of mouse and human cytokines. The technique is based on use of two anti-cytokine monoclonal antibodies (MAbs), each specific for a spatially distinct determinant on the cytokine. One of these is a coating antibody, and the other is a derivatized detecting antibody. A support protocol describes chemical labeling of anti-cytokine monoclonal IgG antibodies with the NIP hapten group to produce the detecting antibody. Another support protocol describes a method for producing horseradish peroxidase (HRPO)-conjugated J4, a rat IgG1 anti-NIP monoclonal antibody that confers the anti-NIP specificity to the HRPO detection system used in the immunoenzymetric assay. The method described in this unit has allowed successful measurement of different cytokines in a wide variety of clinical samples including conditioned medium, serum and plasma, ascites, amniotic fluid, and bronchoalveolar lavage fluid.

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a− DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c− DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcγRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.

IL-18 is critical in eliciting IFN-γ production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (Kd of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-κB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-γ by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.

We have identified a novel member of the calcium-dependent (C-type) lectin family. This molecule, designated DCIR (for dendritic cell (DC) immunoreceptor), is a type II membrane glycoprotein of 237 aa with a single carbohydrate recognition domain (CRD), closest in homology to those of the macrophage lectin and hepatic asialoglycoprotein receptors. The intracellular domain of DCIR contains a consensus immunoreceptor tyrosine-based inhibitory motif. A mouse cDNA, encoding a homologous protein has been identified. Northern blot analysis showed DCIR mRNA to be predominantly transcribed in hematopoietic tissues. The gene encoding human DCIR was localized to chromosome 12p13, in a region close to the NK gene complex. Unlike members of this complex, DCIR displays a typical lectin CRD rather than an NK cell type extracellular domain, and was expressed on DC, monocytes, macrophages, B lymphocytes, and granulocytes, but not detected on NK and T cells. DCIR was strongly expressed by DC derived from blood monocytes cultured with GM-CSF and IL-4. DCIR was mostly expressed by monocyte-related rather than Langerhans cell related DC obtained from CD34+ progenitor cells. Finally, DCIR expression was down-regulated by signals inducing DC maturation such as CD40 ligand, LPS, or TNF-alpha. Thus, DCIR is differentially expressed on DC depending on their origin and stage of maturation/activation. DCIR represents a novel surface molecule expressed by Ag presenting cells, and of potential importance in regulation of DC function.

Optimal T cell activation and expansion require engagement of the TCR plus costimulatory signals delivered through accessory molecules. SLAM (signaling lymphocytic activation molecule), a 70-kDa costimulatory molecule belonging to the Ig superfamily, was defined as a human cell surface molecule that mediated CD28-independent proliferation of human T cells and IFN-gamma production by human Th1 and Th2 clones. In this study, we describe the cloning of mouse SLAM and the production of mAb against it which reveal its expression on primary mouse T and B cells. Mouse SLAM is expressed on highly polarized Th1 and Th2 populations, and is maintained on Th1, but not on Th2 clones. Anti-mouse SLAM mAb augmented IFN-gamma production by Th1 cells and Th1 clones stimulated through the TCR, but did not induce IFN-gamma production by Th2 cells, nor their production of IL-4 or their proliferation. Mouse SLAM is a 75-kDa glycoprotein that upon tyrosine phosphorylation associates with the src homology 2-domain-containing protein tyrosine phosphatase SHP-2, but not SHP-1. Mouse SLAM also associates with the recently described human SLAM-associated protein. These studies may provide new insights into the regulation of Th1 responses.

BALB/c mice develop toxoplasmic encephalitis (TE) but the pathology resolves; in contrast, CBA/Ca mice develop progressive TE. Immunohistochemical analysis revealed peak staining for interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) in lymph nodes of both strains on day 7 of infection, with elevated levels of IFN-gamma and TNF persisting in CBA/Ca mice. TNF was present in the central nervous systems (CNS) of both strains of mice and correlated with severity of pathology. Interleukin (IL)-2 was detected in lymph nodes and CNS of CBA/Ca mice after day 52 of infection and only in the lymph nodes of BALB/c mice between days 14 and 21 after infection. Highest levels of IL-10 were observed in lymph nodes and CNS of infected CBA/Ca mice. Variations in cytokine levels in lymph nodes and CNS of these mouse strains may account for differences in their susceptibility to TE.

OBJECTIVE: Low birth weight remains the leading cause of perinatal morbidity and mortality, but mechanisms mediating impaired fetal growth are poorly understood. To further define the role of abnormal immune activation and suppression in mediating impaired fetal growth, we measured levels of interleukin-10, a potent immunosuppressive cytokine not previously identified in association with pregnancy, in amniotic fluid samples obtained at genetic amniocentesis.STUDY DESIGN: In a case-control study with an enzyme-linked immunoassay we compared amniotic fluid levels of interleukin-10 in midtrimester samples obtained from appropriate-for-gestational age (n = 42) and small-for-gestational-age (n = 24) pregnancies.RESULTS: Interleukin-10 levels in small-for-gestational-age samples were elevated (median 78 pg/ml) compared with levels in control samples (median < 40 pg/ml), p = 0.02. In small-for-gestational-age pregnancies elevated levels were associated with nulliparity, p = 0.003.CONCLUSION: Our data support the role of abnormal immune activation, as opposed to inadequate immune suppression, in mediating impaired fetal growth.

Excessive cytokine expression induced by superantigen may be one aspect of the pathophysiology associated with Gram positive bacteremia. We have undertaken a study of the kinetics of cytokine production in lymph nodes obtained from in vivo Staphylococcus enterotoxin B (SEB) treated animals. This study was designed to evaluate the short term cytokine profile observed using immunohistochemistry (IHC) in BALB/c mice injected intraperitoneally (i.p.). The observed immunohistochemical kinetic profiles were corroborated using reverse transcription-polymerase chain reaction (RT-PCR) RNA analysis. We report here that TNF, IL-2, and IFN-γ are the principal cytokines which were detected within hours of SEB administration, and that other cytokines such as IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF and M-CSF were undetectable. TNF and IL-2 appeared very early following SEB priming, and were observed by 1 h. IFN-γ which appeared later (maximally at 14 h) was produced predominantly by CD8+ cells. In contrast, the TNF and IL-2 were produced primarily by CD4+ cells. Identical results were obtained by IHC and RT-PCR; the kinetics of mRNA expression slightly preceded the appearance of protein. The TNF and IFN-γ staining patterns observed in lymph node sections were indicative of Golgi-localized cytokine. The UL-2 staining pattern observed in lymph node sections was distinctive, covering a significant local area of cells. This local regional concentration of IL-2, which may result from cytokine attached to extracellular binding components, may be an important aspect of the activation phase of a developing immune response. Rapid induction and excessive cytokine production elicited by superantigen in vivo, may ultimately help to explain the shock and death associated with SEB.

Although Th2 helper cell clones produce IL-4 and IL-5, CD4+ T cells taken fresh from lymphoid organs of mice produce IL-2 and some IFN-γ, but not IL-4 or IL-5. The exact parameters that enhance the synthesis or IL-4, IL-5, and particularly IL-10 from resting antigen-specific CD4+ T cells is not yet clear. We therefore examined the kinetics of, and the parameters that affect, the development of IL-4, IL-5, and IL-10 production in bulk populations of antigen-specific human CD4+ T cells. We demonstrated that in vitro stimulation of human peripheral blood lymphocytes with antigen (tetanus toxoid or a vital antigen, Varicella zoster) for several days resulted in the production of IL-2 and IFN-γ, but little or no IL-4 or IL-5. This cytokine profile was observed even when CD4+ T cells from allergic donors were stimulated once in vitro with allergen (rye grass pollen, Lolium perenne I, or dust mite allergen. Dermatophagoides farinae). The observed cytokine profile reflected that of the vast majority of antigen-specific T cells, since we studied the response of bulk populations of CD4+ T cells lather than that of a "selected" number of antigenspecific T cell clones. Furthermore, we observed that human CD4+ T cells from either allergic or nonallergic individuals failed to produce significant quantities of IL-4, IL-5, or IL-10 even after several rounds of stimulation with soluble protein (nonallergen) antigens such as tetanus toxoid or Var. z. However, large quantities of these cytokines were produced when stimulation or the T cells occurred in the presence of rIL-4, but only after two stimulations in vitro with antigen over a period of greater than 7 days. In addition, substantial quantities or IL-4, IL-5, and IL-10 were produced by CD4+ T cells from allergic subjects in the absence of exogenous IL-4, but only after two stimulations in vitro with allergens such as rye grass pollen or dust mite allergen. These results indicate that the development of IL-4 and IL-5 synthesis occurs in peripheral blood CD4+ T cells in a stepwise fashion, first with the production of IL-2 and IFN-γ, and later with the production of IL-4 and IL-5. In addition, these studies are the first to demonstrate that IL-4 upregulates the synthesis of IL-10 in humans and indicate that the synthesis of IL-4, IL-5, and of IL-10 are coordinately regulated in a manner different from that of IL-2 and IFN-γ.

Recent data indicate a major role for IL‐10 in suppressing immune and inflammatory reactions. To date, expression of human IL‐10 has been attributed primarily to helper T lymphocytes, activated monocytes, and neoplastic B cells, and was often found to be associated with IL‐6 expression. In this study we sought to determine whether non‐hematopoietic human tumor cell lines produce IL‐10 and, if so, what is the relationship between IL‐10 and IL‐6. Using ELISA, we determined IL‐10 and IL‐6 levels in culture supernatants of 48 cell lines established from carcinomas of the kidney, colon, breast and pancreas, malignant melanomas and neuroblastomas. IL‐6 protein was secreted by 28 of the tumor ceil lines; IL‐10 was measurable in 15 cell lines. IL‐6 secretion was maximal and most frequent in renal‐cancer cell lines, while IL‐10 production was found to be highest and most common among cell lines derived from colon carcinomas. IL‐10 in conditioned medium of one of the colon carcinoma cell lines (CCL222) was bio‐active, as demonstrated in the mouse MC/9 mast‐cell‐line assay and in human mixed‐lymphocyte reactions. In both assays, IL‐10 bio‐activity was neutralized by an anti‐IL‐10 monoclonal antibody. Expression of IL‐6 and IL‐10 was confirmed by RNA analysis using message amplification by PCR and sequencing of amplified cDNA. LPS, IL‐1 alpha, and TNF‐alpha strongly enhanced the release of IL‐6 by RCC cells, but only marginally affected IL‐10 production in colon‐carcinoma cells. IL‐10 secretion by colon‐carcinoma cells was moderately stimulated by IFN‐gamma and IL‐4. Dexamethasone suppressed the release of IL‐6, but had no inhibitory effect on IL‐10 secretion. Our results demonstrate that tumor cell lines established from certain types of human carcinomas are capable of expressing and releasing IL‐6 and/or IL‐10, suggesting a role of these cytokines in solid‐tumor development and anti‐tumor immunity. © 1993 Wiley‐Liss, Inc.

Levels of cytokine mRNA were studied in the central nervous system (CNS) of SCID mice infected with Toxoplasma gondii. This infection led to 100% mortality by day 23 postinfection. Inflammation was observed in the lungs on day 7 and in the heart, liver, and kidneys on days 14 and 18 of infection. In the CNS, necrotic, acellular lesions that contained numerous parasites, accompanied by a localized astrocyte activation, were evident on day 14. Polymerase chain reaction-assisted amplification of RNA revealed that, although transcripts for interleukin-1 alpha (IL-1 alpha) and IL-1 beta were present in the brains of uninfected mice, increased levels of these transcripts were detected on day 7 of infection. Transcripts for macrophage inflammatory protein 1 and transforming growth factor beta were also detected in brains of infected mice at this time point. On days 14 and 18, levels of these transcripts had increased and transcripts for IL-6, IL-10, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were also detected. Transcripts for IL-2 or IL-4 were not detected at any of the time points. Detection of locally produced cytokine transcripts may reflect involvement of the cytokines in the immunopathogenesis of this infection or involvement in mediating antitoxoplasma activity. To assess the possible role of endogenous IFN-gamma, TNF-alpha, IL-10, IL-6, and GM-CSF, cytokine-neutralizing monoclonal antibodies were administered to infected SCID mice. Neutralization of IFN-gamma or TNF-alpha led to earlier mortality than that in controls. In contrast, treatment with antibody to IL-10 and IL-6 increased survival time. Treatment with anti-GM-CSF did not alter the time to death. These results indicate that TNF-alpha and IFN-gamma are both involved in T-cell-independent mechanisms of resistance to T. gondii in SCID mice and that IL-10 and IL-6 may downregulate the immune response to this pathogen.

Typically, ovarian cancer remains restricted to the peritoneal cavity. Because of this unique localization, the study of ovarian cancer is particularly suitable for immune analysis and for the development of immunotherapy. Here we report that peritoneal fluid from patients with ovarian or other intra-abdominal cancers contained significantly elevated levels of interleukin 10 (IL-10) (542 +/- 77 pg/ml, N = 35), compared with peritoneal fluid from patients with benign gynecological conditions (34.2 +/- 7.5 pg/ml, N = 63) (P < 0.001). Peritoneal fluid IL-10 levels did not correlate with histology, tumor stage, grade, or prognosis. IL-10 levels were also elevated in the serum of patients with intra-abdominal cancer (1353 +/- 906, N = 8). Established ovarian cancer cell lines (N = 5) did not produce any detectable IL-10. Investigation of the cell surface phenotype of the cells in the peritoneal cavity indicated the presence of significant amounts of activated immune cells. The presence of cytokines such as IL-10 in the peritoneal cavity of ovarian cancer bearing patients could be important in the growth and development of cancer, more specifically, in relation to host immune responsiveness.

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.

SUMMARYTwo subsets of differentiated murine helper T cells, Th1 and Th2, based on secretion products in response to antigen have been described (Cher & Mosmann 1987, Coffman et al. 1988, Lopez et al. 1988, Paliard et al. 1988, Patel et al. 1988, Mosmann & Coffman 1989). To analyse immunological function of antigen‐specific CD4+ T cells in human schistosomiasis, we produced schistosomal egg antigenspecific T cell clones from a former patient. We identified four different types of CD4+ T cell clones by analysis of cytokine production. Two of the four types of the clones corresponded to murine Th1 or Th2 subsets; a third type was of the Th0 subset (Th1 + 2) and a fourth type produced IL‐5 dissociated from IL‐4. Analysis of the antigen(s) recognized by these T cell clones showed that all of the clones proliferated in response to soluble egg antigen(s) (SEA) found within a pl fraction whose pH was 5·2. T cell Western blot analysis of the stimulatory pl fraction demonstrated that the apparent Mr of the relevant antigens recognized by the clones were 38 kDafor the Th2 homologue, and 45–55 kDa for the Th1 homologue.