John S. Abrams

Novel p19 Protein Engages IL-12p40 to Form a Cytokine, IL-23, with Biological Activities Similar as Well as Distinct from IL-12

A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.

Strategies of Anti-Cytokine Monoclonal Antibody Development: Immunoassay of IL-10 and IL-5 in Clinical Samples

The identification of cytokines, a class of intermediate molecular weight, intercellular regulatory proteins, and an understanding of their function has been facilitated tremendously by the use of recombinant DNA technology. Following closely upon these developments has come the ability to detect and quantify cytokines in clinical and biological samples, in large part made possible by the use of specific and sensitive immunoassays using anti-cytokine antibodies. Cytokine detection with antibody-based techniques is particularly useful because so many of these factors have overlapping, parallel, or counter-regulatory bioactivities and it is extremely difficult to distinguish and measure them in complex mixtures on the basis of biological activity alone, Therefore, both neutralizing antibodies in bioassay systems and cytokine immunoassays of complex samples are required (Mosmann & Fong 1989). In the first part of this review, we outline the general strategy we have followed for preparing a large panel of anti-mouse and human cytokine monoclonal antibodies (mAb) and setting up cytokine immunoassays. While there is a general consensus that many of the frequently produced cytokines such as IL-1, TNF, IFN-y, IL-6, and IL-8 may be general inflammatory mediators, see for example (Dunn 1991), definitive links between the detection of specific cytokine patterns and the pathogenesis of particular diseases are in the early stages of being understood. The eventual elucidation of such cause-and-

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.

Interleukin-6 Production by Schwann Cells and Induction in Sciatic Nerve Injury

Abstract: Interleukin‐6 (IL‐6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell‐derived IL‐6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL‐6. Cocultured iSC transcribe IL‐6 message as confirmed by northern analysis. Stimuli that induce IL‐6 production in the hematopoietic lineage induced transcription and production of IL‐6 by iSC cells. Lipopolysaccharide, tumor necrosis factor‐α, IL‐1α, IL‐6, and serum withdrawal induced iSC cell IL‐6 mRNA. The kinetics of IL‐6 production was confirmed in the mouse IL‐6‐dependent B9 bioassay and that activity could be neutralized with antisera to IL‐6. Expression of both the IL‐6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL‐6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL‐6 message is induced. IL‐6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL‐6 in response to PC12 cell coculture and to stimuli that induce IL‐6 production in the hematopoietic lineage. The induction of IL‐6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.

Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2)

We report the identification and characterization of a novel CC chemokine designated CCL28 and its receptor CCR10, known previously as orphan G-protein-coupled receptor GPR2. Human and mouse CCL28 share 83% identity at the amino acid and 76% at the nucleic acid levels. We also identified the mouse homologues of CCL28 and of CCR10, which map to mouse chromosomes 13 and 11, respectively. CCL28 is expressed in a variety of human and mouse tissues, and it appears to be predominantly produced by epithelial cells. Both human and mouse CCL28 induce calcium mobilization in human and mouse CCR10-expressing transfectants. CCL28 desensitized the calcium mobilization induced in CCR10 transfectants by CCL27, indicating that these chemokines share this new chemokine receptor. In vitro, recombinant human CCL28 displays chemotactic activity for resting CD4 or CD8 T cells.

Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining.

Production of IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, GM-CSF (granulocyte-macrophage colony stimulating factor) and macrophage colony stimulating factor by murine peripheral blood and spleen cells was analyzed following primary and secondary mitogen stimulation in vitro. Individual cytokine producing cells were detected by an intracytoplasmic staining technique. Cytokine production in cells from peripheral blood and spleen was comparable and more rapidly induced by calcium ionophore and phorbol 12-myristate 13-acetate than by concanavaling A. Restimulation in vitro induced both a swift production of cytokines and, for some cytokines, higher frequencies of producing cells. This was especially evident for IL-10 secreting cells, which increased 30-80 times in secondary responses. These analyses using the dual approaches of immunoenzymetric and fluorescent immunohistochemical techniques provide important evidence that cytokine induction kinetics can differ following primary or secondary stimulation.

Cytokine Networking in Lungs of Immunocompetent Mice in Response to Inhaled Aspergillus fumigatus

ABSTRACT Cytokine networking in the lung in response to inhaledAspergillus fumigatus was assessed using a murine model of primary pulmonary aspergillosis in immunocompetent Crl:CF-1 mice. Inhalation of virulent A. fumigatus (6 × 106 CFU) resulted in the induction of interleukin 18 (IL-18), tumor necrosis factor alpha (TNF-α), IL-12, and gamma interferon (IFN-γ) protein in bronchoalveolar lavage fluid and/or lung tissue. Induction of immunoreactive IL-18 preceded induction of TNF-α protein, which preceded induction of immunoreactive IL-12 and IFN-γ. Real-time reverse transcriptase (RT) PCR analysis of infected lung tissue demonstrated that induction of IL-18 protein also preceded induction of pulmonary TNF-α, IL-12, and IFN-γ mRNAs. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to the IL-18 receptor (anti-IL-18R MAb), TNF-α (anti-TNF-α MAb), IL-12 (anti-IL-12 MAb), and/or IFN-γ (anti-IFN-γ MAb), and effects on intrapulmonary cytokine activity and growth of A. fumigatuswere assessed in infected lung homogenates. Simultaneous neutralization of IL-12 and IL-18 resulted in decreased levels of immunoreactive TNF-α, while neutralization of IL-18, TNF-α, or IL-12 alone or of IL-18 and IL-12 together resulted in decreased levels of immunoreactive IFN-γ. Simultaneous neutralization of IL-12 and IL-18 or neutralization of TNF-α alone or in combination with IL-12, IL-18, or IFN-γ also resulted in a significant increase inA. fumigatus CFU in lung tissue. Taken together, these results demonstrate that endogenous IL-18, IL-12, and TNF-α, through their modulatory effects on both intrapulmonary cytokine activity and growth of A. fumigatus, play key roles in host defense against primary pulmonary aspergillosis.

FDF03, a Novel Inhibitory Receptor of the Immunoglobulin Superfamily, Is Expressed by Human Dendritic and Myeloid Cells

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.

CCL18/DC-CK-1/PARC up-regulation in hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a lung inflammatory disorder characterized by accumulation of T lymphocytes. However, the mechanisms implicated in this process remain undefined. We examined the expression of dendritic cell (DC)‐derived CC chemokine 1 (CK1)/CCL18, a chemokine putatively involved in naive T cell recruitment, in lungs from 10 patients with HP, 9 patients with idiopathic pulmonary fibrosis (IPF), and 20 healthy lungs. CCL18 was measured by real‐time quantitative PCR and localized in lungs by in situ hybridization and immunohistochemistry. CCL18 expression was significantly increased in lungs affected by HP in comparison with lungs affected by IPF (2,085±393 vs. 1,023±110; P<0.05) and controls (2,085±393 vs. 467±94; P<0.01). Macrophages, DCs, and alveolar epithelial cells were the main sources of CCL18. There was a direct correlation between the levels of tissue CCL18 and the number of lymphocytes in the bronchoalveolar lavage fluids. High levels of CCL18 were detected in the subacute rather than the chronic phase of HP. These findings suggest a role for CCL18 in the pathogenesis of HP.

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.