Granulocyte/macrophage colony-stimulating factor and interleukin 3 release from human peripheral blood eosinophils and neutrophils.

Human peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin. The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A. The survival-enhancing activity was completely abolished by treatment with anti-interleukin 3 (IL-3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies. Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL-3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced IL-3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms.

Regulation of parasite-induced eosinophilia: selectively increased interleukin 5 production in helminth-infected patients.

Production of the eosinophilogenic cytokines interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and IL-5 by mitogen-stimulated peripheral blood mononuclear cells was compared between 11 noneosinophilic individuals and seven patients with helminth-induced eosinophilia. Both the kinetics and quantities of IL-3 and GM-CSF were similar in the two groups. In contrast, IL-5 production at both the protein and the mRNA level was markedly greater in the eosinophilic patients, an observation suggesting that IL-5 may be particularly important in mediating the selective eosinophilia seen in filarial and other helminth infections.

Induction of human IgE synthesis by CD4+ T cell clones. Requirement for interleukin 4 and low molecular weight B cell growth factor.

We have analyzed in detail the precise requirements for the induction of human IgE synthesis using several experimental approaches with purified B cells and well-characterized alloantigen-specific CD4+ T cell clones expressing different profiles of lymphokine secretion. Using these clones under cognate conditions in which the B cells expressed alloantigens recognized by the cloned T cells, we have confirmed that IL-4 is required for the induction of IgE synthesis, but we have clearly demonstrated that IL-4 by itself is not sufficient. With several cloned CD4+ T cell lines, including an IL-4-producing clone that could not induce IgE synthesis, and cloned T cells pretreated with cyclosporin A to inhibit lymphokine synthesis, we showed that Th cell-B cell interactions are necessary for IgE synthesis, and that low molecular weight B cell growth factor (LMW-BCGF) and IL-4, in combination, are lymphokines of major importance in the induction of IgE synthesis. Together our results indicate that optimal induction of an IgE-specific response requires the exposure of B cells to a particular complex of signals that include (a) a signal(s) involving Th-B cell interaction that primes B cells to receive additional signals from soluble lymphokines, (b) a specific B cell proliferative signal provided by LMW-BCGF, and (c) a specific B cell differentiation signal provided by IL-4.

A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.

Cholera toxin (CT) has been shown to induce stem cell factor (SCF) production in mouse ligated intestinal loops. Further, SCF interaction(s) with its receptor (c-kit) was shown to be important for the intestinal tract secretory response after CT exposure. In this study, we have investigated whether SCF production is induced in the intestinal tract after exposure to Salmonella typhimurium and whether this production could be an important intestinal tract response to Salmonella infection. Using a mouse ligated intestinal loop model, increased levels of SCF mRNA were detected at 2-4 h post-Salmonella challenge. Intestinal fluid obtained from Salmonella-challenged loops contained high levels of SCF by ELISA. Human and murine intestinal epithelial cell lines were also shown to have increased levels of SCF mRNA after exposure to Salmonella. Inhibition of Salmonella invasion of epithelial cells was shown to be one potentially important role for SCF:c-kit interactions in host defense to Salmonella infection. Pretreatment of human or murine intestinal cell lines with SCF resulted in a cellular state that was resistant to Salmonella invasion. Finally, mice having mutations in the white spotting (W) locus, which encodes the SCF-receptor (c-kit), were significantly more susceptible to oral Salmonella challenge than their control littermates. Taken together, the above results suggest that an important intestinal tract response to Salmonella infection is an enhanced production of SCF and its subsequent interactions with c-kit.

Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10.

The mechanism by which the mammalian mother accepts the implanting fetus as an allograft remains unexplained, but is likely to be the result of a combination of factors. Mononuclear cytotrophoblasts, the specialized fetal cells of the placenta that invade the uterus, play an important role. These cells express HLA-G, an unusual major histocompatibility complex class I-B molecule, and secrete cytokines and pregnancy-specific proteins that can regulate immune function. We investigated whether cytotrophoblasts secrete interleukin 10 (IL-10), a cytokine that potently inhibits alloresponses in mixed lymphocyte reactions. Cytotrophoblasts from all stages of pregnancy produced IL-10 in vitro, but neither placental fibroblasts nor choriocarcinoma (malignant trophoblast) cell lines did so. Spontaneous IL-10 production averaged 650, 853, and 992 pg/10(6) cells in the first, second, and third trimesters of pregnancy, respectively. IL-10 secretion dropped approximately 10-fold after the first 24 h of culture, and was paralleled by a decrease in messenger RNA. IL-10 messenger RNA was detected in biopsies of the placenta and the portion of the uterus that contains invasive cytotrophoblasts, suggesting that this cytokine is also produced in vivo. IL-10 secreted by cytotrophoblasts in vitro is bioactive, as determined by its ability to suppress interferon gamma production in an allogeneic mixed lymphocyte reaction. We conclude that human cytotrophoblast IL-10 may be an important factor that contributes to maternal tolerance of the allogeneic fetus.

A critical role for interleukin 18 in primary and memory effector responses to Listeria monocytogenes that extends beyond its effects on Interferon gamma production.

The stimulation of interferon (IFN)-gamma by interleukin (IL)-12 has been shown to provide protection from intracellular pathogens such as Listeria monocytogenes. Tumor necrosis factor (TNF) is also a major player in the resolution of Listeria infections and is suggested to have more global effects than can be explained by the induction of IFN-gamma alone. Since IL-18 synergizes with IL-12 to induce IFN-gamma production by natural killer and T helper (Th)1 cells, we determined its role in responses to Listeria. IL-18 appeared to be even more potent than either IL-12 or IFN-gamma for protection against this pathogen and IL-18 enhanced bacterial clearance in the complete absence of IFN-gamma. Indeed IL-18 was comparable to TNF in its ability to resolve the infection and showed a lowered protective capacity in the absence of TNF. Moreover, IL-18 induced macrophages to secrete both TNF and nitric oxide after a Listeria infection. IL-18 was also essential for optimal IFN-gamma production by antigen-specific T cells. Therefore, IL-18 operates via its effects on both the innate immune response, including macrophages, as well as on Th1 cells, to protect against Listeria.

Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)-G bind to peripheral blood myelomonocytic cells.

The nonclassical MHC class I molecule human histocompatibility leukocyte antigen (HLA)-G is selectively expressed on fetal trophoblast tissue at the maternal-fetal interface in pregnancy. It has long been suggested that HLA-G may inhibit maternal natural killer (NK) cells through interaction with particular NK cell receptors (KIRs). To investigate interactions of HLA-G, we constructed phycoerythrin-labeled tetrameric complexes of HLA-G refolded with a self-peptide. These HLA-G tetramers failed to bind to NK cells and cells transfected with CD94/NKG2 and killer immunoglobulin-like NK receptors. In contrast, HLA-G tetramers did bind to peripheral blood monocytes, staining a CD16(+)CD14(mid) subset with greater intensity. On transfectants, HLA-G tetramers bound to inhibitory immunoglobulin-like transcript (ILT)2 and ILT4 receptors. However, staining in the presence of antibodies reactive with ILT receptors revealed that the interaction of HLA-G tetramers with blood monocytes was largely due to binding to ILT4. These results suggest that the primary role of HLA-G may be the modulation of myelomonocytic cell behavior in pregnancy.

COOH-terminal-modified interleukin-3 is retained intracellularly and stimulates autocrine growth.

Autocrine growth due to dysregulated growth factor production may have a role in the development of neoplasia. Whether autocrine growth is stimulated by growth factor secretion in an autocrine loop or by intracellular binding of the growth factor to a receptor has been unclear. The carboxyl-terminus coding sequence for murine interleukin-3 (IL-3) was extended with an oligonucleotide encoding a four-amino acid endoplasmic reticulum retention signal. IL-3-dependent hematopoietic cells became growth factor-independent when the modified IL-3 gene was introduced by retroviral gene transfer, despite lack of secretion of the modified IL-3. Hence autocrine growth can occur as a result of the intracellular action of a growth factor and this mechanism may be important in neoplastic and normal cells.

Activation of mouse T-helper cells induces abundant preproenkephalin mRNA synthesis.

Antigenic or mitogenic stimulation of T cells induces the secretion of an array of protein hormones that regulate immune responses. Molecular cloning has contributed strongly to our present understanding of the nature of this regulation. A complementary DNA (cDNA) library prepared from a cloned concanavalin A-activated mouse T-helper cell line was screened for abundant and induction-specific cDNA's. One such randomly chosen cDNA was found to encode mouse preproenkephalin messenger RNA (mRNA). Preproenkephalin mRNA represented about 0.4 percent of the mRNA in the activated cell line but was absent in resting cells of this line. Other induced T-helper cell lines have 0.1 to 0.5 percent of their mRNA as preproenkephalin mRNA. Induced T-helper cell culture supernatants have [Met]enkephalin-immunoreactive material. The production by activated T cells of a peptide neurotransmitter identifies a signal that can potentially permit T cells to modulate the nervous system.

Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones.

Functional subsets of human T cells were delineated by analyzing patterns of lymphokines produced by clones from individuals with leprosy and by T cell clones of known function. CD4 clones from individuals with strong cell-mediated immunity produced predominantly interferon-gamma, whereas those clones that enhanced antibody formation produced interleukin-4. CD8 cytotoxic T cells secreted interferon-gamma. Interleukin-4 was produced by CD8 T suppressor clones from immunologically unresponsive individuals with leprosy and was found to be necessary for suppression in vitro. Both the classic reciprocal relation between antibody formation and cell-mediated immunity and resistance or susceptibility to certain infections may be explained by T cell subsets differing in patterns of lymphokine production.

Increased osteoclast development after estrogen loss: mediation by interleukin-6.

Osteoclasts, the cells that resorb bone, develop from hematopoietic precursors of the bone marrow under the control of factors produced in their microenvironment. The cytokine interleukin-6 can promote hematopoiesis and osteoclastogenesis. Interleukin-6 production by bone and marrow stromal cells is suppressed by 17 beta-estradiol in vitro. In mice, estrogen loss (ovariectomy) increased the number of colony-forming units for granulocytes and macrophages, enhanced osteoclast development in ex vivo cultures of marrow, and increased the number of osteoclasts in trabecular bone. These changes were prevented by 17 beta-estradiol or an antibody to interleukin-6. Thus, estrogen loss results in an interleukin-6-mediated stimulation of osteoclastogenesis, which suggests a mechanism for the increased bone resorption in postmenopausal osteoporosis.

Intent-to-treat analysis of the placebo-controlled trial of letrozole for extended adjuvant therapy in early breast cancer: NCIC CTG MA.17.

BACKGROUND: MA.17 evaluated letrozole or placebo after 5 years of tamoxifen and showed significant improvement in disease-free survival (DFS) for letrozole [hazard ratio (HR) 0.57, P = 0.00008]. The trial was unblinded and placebo patients were offered letrozole. PATIENTS AND METHODS: An intent-to-treat analysis of all outcomes, before and after unblinding, on the basis of the original randomization was carried out. RESULTS: In all, 5187 patients were randomly allocated to the study at baseline and, at unblinding, 1579 (66%) of 2383 placebo patients accepted letrozole. At median follow-up of 64 months (range 16-95), 399 recurrences or contralateral breast cancers (CLBCs) (164 letrozole and 235 placebo) occurred. Four-year DFS was 94.3% (letrozole) and 91.4% (placebo) [HR 0.68, 95% confidence interval (CI) 0.55-0.83, P = 0.0001] and showed superiority for letrozole in both node-positive and -negative patients. Corresponding 4-year distant DFS was 96.3% and 94.9% (HR 0.80, 95% CI 0.62-1.03, P = 0.082). Four-year overall survival was 95.1% for both groups. The annual rate of CLBC was 0.28% for letrozole and 0.46% for placebo patients (HR 0.61, 95% CI 0.39-0.97, P = 0.033). CONCLUSIONS: Patients originally randomly assigned to receive letrozole within 3 months of stopping tamoxifen did better than placebo patients in DFS and CLBC, despite 66% of placebo patients taking letrozole after unblinding.

Regulation of immunoglobulin (Ig)E synthesis in the hyper-IgE syndrome.

The hyper-IgE (HIE) syndrome is characterized by high IgE serum levels, chronic dermatitis, and recurrent infections. The mechanisms responsible for hyperproduction of IgE in HIE patients are presently unknown. We investigated whether spontaneous in vitro IgE synthesis by PBMC from seven HIE patients was sensitive to signals (cell adhesion, T/B cell cognate interaction and lymphokines: IL-4, IL-6, and IFN-gamma) known to regulate IgE induction in normals. Our results show that, unlike IL-4 dependent IgE synthesis induced in normals, spontaneous IgE production by PBMC from HIE patients was not blocked by monoclonal antibodies to CD2, CD4, CD3, and MHC class II antigens. Furthermore, antibodies to IL-4 and IL-6 did not significantly suppress IgE production. IFN-gamma had no significant effects on spontaneous in vitro IgE synthesis. To test whether an imbalance in lymphokine production might underlie hyperproduction of IgE in HIE patients, mitogen-induced secretion of IL-4 and IFN-gamma by PBMC was assessed. No significant difference was detected between HIE patients and normal controls. Thus, ongoing IgE synthesis in the HIE syndrome is largely independent of cell-cell interactions and endogenous lymphokines, and is due to a terminally differentiated B cell population, no longer sensitive to regulatory signals.

Interleukin-5 and the posttreatment eosinophilia in patients with onchocerciasis.

To understand the role of the eosinophilopoietic cytokine IL-5 in humans, the posttreatment eosinophilic response in a group of microfilaria (mf)-positive patients with onchocerciasis (n = 10) was examined before and after treatment with diethylcarbamazine (6 mg/kg for 7 d). Sequential blood samples were assessed at 24 and 1 h before treatment (baseline values), then at frequent intervals over the next 14 d. Symptom scores, skin microfilariae (mf), and peripheral blood eosinophil counts were recorded as a function of time after treatment, and serum levels of IL-5 were quantitated by a highly sensitive (sensitivity greater than or equal to 20 pg/ml) monoclonal-based ELISA. Pretreatment eosinophil counts ranged from 240 to 1,186 eosinophils/microliter (geometric mean, 675), and the mf counts from 10 to 218 per mg skin (geometric mean, 79). After an initial decline in the peripheral eosinophil count to 28 +/- 8% of pretreatment levels at 8 h after beginning treatment, the eosinophil counts steadily increased over the next 2 wk, reaching a maximum at 14 d (257 +/- 38% of pretreatment levels). Serum levels of IL-5 rose sharply from pretreatment levels to a peak of 70.5 +/- 11 pg/ml by 24 h after treatment. Serum IL-5 remained elevated over the next 2-3 d and declined toward baseline by approximately 6 d after treatment, at which time the eosinophil levels were steadily increasing. IL-3 and granulocyte macrophage colony-stimulating factor, two other cytokines implicated in eosinophilopoeisis, were not detectable in the serum at any time before or after treatment. The rise in serum IL-5 before the posttreatment eosinophilia seen in this group of patients with onchocerciasis demonstrates a temporal relationship between IL-5 and the subsequent development of eosinophilia and implicates IL-5 as an important mediator of eosinophilia in humans.

Local cytokine production in a murine model of Escherichia coli pyelonephritis.

Cytokines may play an important role in the regulation of host defense against local bacterial infections. We have evaluated the local production of cytokines in a BALB/c mouse model of Escherichia coli pyelonephritis. Kidneys, draining lymph nodes, and spleens, were harvested at specific time intervals after bladder inoculation with E. coli corresponding to the stages of renal infection, infiltration, and bacterial clearance seen in this model. The presence of messenger RNA for specific cytokines (interleukins 1 through 6, chemotactic factors, granulocyte and granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF alpha) and beta, IFN gamma, transforming growth factor (TGF beta), and cytokine synthesis inhibitory factor (CSIF)/IL-10) was determined by polymerase chain reaction (PCR) amplification of reverse transcribed RNA. We have demonstrated mRNA encoding IL-1, IL-6, G-CSF, GM-CSF, TNF alpha, H400 (a protein homologous to a family of chemotactic factors and identical to MIP-1 beta), and CSIF/IL-10 in the kidney at 12 h and 1, 2, and 3 d after bacterial challenge. No signal was seen in normal animals or in mice after 5 d. This pattern of cytokine expression was observed only in renal tissues suggesting a localized response. IL-6 was present in the urine at 4 h with rapid resolution to baseline levels by 24 to 48 h. In contrast, IL-6 was not usually detectable in the serum. TNF alpha was not detectable in the serum or urine during the course of the infection. By immunohistochemical staining of kidney sections we have shown that IL-6 is produced predominantly by mesangial cells rather than by the inflammatory infiltrate. This study provides additional evidence utilizing novel techniques that specific cytokines are produced locally in response to bacterial infections. The time course of production demonstrated in this model supports the important role of cytokines in natural host resistance to local infection.

Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset.

The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.

Potential role for interleukin-10 in the immunosuppression associated with kala azar.

Patients with acute kala azar are generally nonreactive in a number of immunologic assays, including T cell proliferation and generation of macrophage-activating cytokines, principally IFN-gamma, in response to leishmania antigens in vitro. To test for potential immunosuppressive factors, a series of T cell lines and clones were established from patients with acute kala azar, from patients after chemotherapy for kala azar, and from skin test-positive adults from the same endemic region. Although CD4+ T cell lines and clones could be readily established from the skin test-positive adults, lines and clones from acute or treated patients were heavily biased in expression of CD8+. The CD8+ cells from acute patients did not themselves release cytokines in response to leishmania antigens in vitro, but markedly affected the cytokine profile of peripheral blood mononuclear cells isolated 1 yr later after recovery. Addition of the CD8+ cells caused inhibition of lymphoproliferation and IFN-gamma release, with augmentation of IL-6 and IL-10 release. The inhibitory effects of the CD8+ cells could be partially abrogated by antibodies to IL-10 but not by antibodies to IL-4. Analysis of four patients with acute kala azar demonstrated release of IL-10 that could not be demonstrated in supernatants from asymptomatic skin test-positive individuals. Generation of IL-10 may contribute to the profound suppression of IFN-gamma release that occurs during kala azar due to Leishmania chagasi.

Mechanism of autocrine stimulation in hematopoietic cells producing interleukin-3 after retrovirus-mediated gene transfer.

Endogenous expression of the interleukin-3 (IL3) gene introduced with a retrovirus vector renders hematopoietic cells autonomous of exogenous growth factor. To investigate the mechanism of autocrine stimulation, 25 clones were isolated after retrovirus transduction of IL3 into 32D-cl23 or FDC-P1 cells. Medium conditioned by these autonomous IL3-producing clones supported the growth of factor-dependent 32D cells. Although there was a severalfold variation in the amount of IL3 secreted (some clones secreted barely detectable levels), the doubling time of each clone in the absence of added IL3 was identical to that of the parental cell line maximally stimulated by exogenous IL3. Concentrated monoclonal and polyclonal antibodies, both highly effective in neutralizing exogenous IL3, were assayed for ability to inhibit autocrine growth. Minimal inhibitory effects were observed only on washed autocrine clones secreting low levels of IL3. To test the activity of cytoplasmically synthesized IL3, the nucleotides encoding the signal sequence of IL3 were deleted and replaced with an in-frame ATG in the context of a consensus translation initiation sequence. Ten 32D clones expressing this restructured IL3 genome were obtained. Despite the presence of biologically active IL3 in cell lysates, all clones remained dependent on exogenous IL3, with the same dose-response as that found for 32D cells. Our data are most compatible with a mechanism whereby endogenously produced IL3 interacts with its receptor prior to surface display.

National Cancer Institute Formulary: A Public-Private Partnership Providing Investigators Access to Investigational Anticancer Agents.

As part of the White House Cancer Moonshot Initiative, the National Cancer Institute (NCI) has developed a drug formulary to provide investigational anticancer agents to the extramural research community. This article describes how the NCI Formulary functions, how researchers may apply for access to drugs in the formulary, and the NCI's initial goals for formulary participation. Approved investigators may apply for access to formulary agents at: https://nciformulary.cancer.gov.