Inhibition of cyclooxygenase-2 prevents adverse effects induced by phosphodiesterase type 4 inhibitors in rats.

BACKGROUND AND PURPOSE: Phosphodiesterase type 4 (PDE4) inhibitors such as roflumilast are currently being developed as anti-inflammatory treatments for chronic airway disorders. However, high doses of PDE4 inhibitors have also been linked to several side effects in different animal species, including pro-inflammatory effects in the rat. Here, we analysed PDE4-related toxicological findings in a rat model and how these side effects might be therapeutically prevented. EXPERIMENTAL APPROACH: Wistar rats were treated orally once daily with 10 mg·kg⁻¹ roflumilast for 4 days. Macroscopic changes were monitored throughout the study and further parameters were analysed at the end of the experiment on day 5. In addition, the effects of concomitant treatment with cyclooxygenase (COX) inhibitors were assessed. KEY RESULTS: Supratherapeutical treatment with roflumilast induced marked body and spleen weight loss, diarrhea, increased secretory activity of the harderian glands, leukocytosis, increased serum cytokine-induced neutrophil chemoattractant-1 (CINC-1) levels, and histopathological changes in thymus, spleen, mesentery and mesenteric lymph nodes. All these toxicological findings could be prevented by the non-steroidal anti-inflammatory drug (NSAID) and non-selective COX inhibitor, diclofenac, given orally. Similar protective effects could be achieved by the COX-2 selective inhibitor lumiracoxib, whereas the COX-1 selective inhibitor SC-560 was generally not effective. CONCLUSIONS AND IMPLICATIONS: Treatment with an NSAID inhibiting COX-2 prevents the major effects found after subchronic overdosing with the PDE4-specific inhibitor roflumilast. If this effect translates into humans, such combined treatment may increase the therapeutic window of PDE4 inhibitors, currently under clinical development.

Molecular cloning of a macrophage-derived, interferon-inducible secreted immunoglobulin-binding protein.

We describe the molecular cloning of a 1803-bp cDNA coding for a product termed interferon-induced immunoglobulin-binding protein (IIBP) from a library of IFN-alpha-induced primary bone marrow macrophages. The open reading frame encodes a protein of 26-kDa containing two immunoglobulin-like and one Fc receptor-like domain. Due to the constitutive release of low amounts of IFN-beta, the IIBP mRNA is already present in macrophage-colony-stimulating factor-cultured macrophages. Its expression could be blocked in the presence of either anti-IFN-beta or interleukin-4, which down-regulates the endogenous IFN-beta production. Upon addition of rIFN-alpha4 a 3-5-fold superinduction of IIBP mRNA was observed. Rat monoclonal antibodies detected a protein of the predicted size exclusively in cell culture supernatants of primary bone marrow macrophages and a B-cell line. In immunoprecipitation experiments an unknown 30-kDa protein co-precipitated. The secreted IIBP showed considerable binding to nonspecific rat IgG2a and could be precipitated using mouse IgG2a, IgG2b and IgG3 antibodies of irrelevant specificities, indicating that this gene product is a novel secreted immunoglobulin-binding protein with a new IgG isotype binding pattern that differs to that of known Fc receptors.

The role of peptide presentation in the physiological function of HLA-G.

The HLA-G gene gives rise to six differently spliced mRNAs. The membrane bound HLA-G1 molecule containing all three extracellular domains presents peptides that follow motif requirements similar to those of classical HLA class I molecules. This isoform is also capable of inhibiting Natural Killer (NK) cells, but is only efficiently transported to the cell surface when peptides are provided in the endoplasmic reticulum. In the absence of sufficient peptide supply to the ER a small molecule of 18-kDa is transported to the cell surface in HLA-G transfectants of LCL721.221 cells. HLA-G transfectants with impaired ER peptide supply are nevertheless protected from NK lysis.

Cell lines transfected with the TAP inhibitor ICP47 allow testing peptide binding to a variety of HLA class I molecules.

The immediate early protein ICP47 of the Herpes simplex virus is known to block the human transporter associated with antigen processing (TAP), thereby creating a TAP-deficient phenotype in any human cell transfected with the corresponding cDNA. Exploiting this inhibitory activity, we constructed a selection of human cell lines each co-expressing one of the cDNAs of human leukocyte antigen (HLA) class I alleles HLA-A*1101, A24, A*3101, A*6601, B8 and B*1516, and the cDNA encoding the ICP47 molecule. The cell lines generated showed diminished HLA class I surface expression and the inhibition of the TAP function was confirmed in peptide translocation assays. The addition of specific exogenous peptide ligands restored the expression of the corresponding HLA class I molecules. Thus, the ICP47 transfectants provide us with a tool to closely examine peptide-HLA class I interactions, to confirm HLA class I ligand motifs and to test peptides predicted to bind.

Interferon-induced expression of If-1h and If-1l alleles in Newcastle disease virus-infected mouse macrophages is associated with specific differences in viral gene transcription.

We have studied the expression of cytokines and viral genes induced by Newcastle disease virus (NDV) and Sendai virus in bone marrow-derived macrophages (BMM) and lymphocytes from C57BL/6 mice and the congenic line B6.C-H-28c. These mice carry the loci If-1h (high) or If-1l (low), respectively, that are responsible for up to tenfold differences in the interferon (IFN)-alpha, IFN-beta, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) response to NDV but not to Sendai virus. Only BMM but not spleen lymphocytes showed allele-specific differences in NDV-induced cytokine levels, indicating cell-specific If-1 expression. The If-1 locus harbors IFN-inducible gene(s) whose expression is prevented in the presence of cycloheximide. Our data provide evidence that the If-1l allele acts by specifically suppressing the cytokine response to NDV. Cytokine production was dependent on infectious virions, and kinetic analyses revealed a close correlation between the amount of viral transcripts and individual cytokine mRNA. BMM from lf-1l mice strongly restricted transcription of the NDV nucleoprotein (NP) gene, whereas BMM from If-1h mice supported NP transcription. Following treatment with IL-4, which inhibited constitutive IFN-beta gene expression, however, If-1l BMM became highly permissive for transcription of the viral NP gene and released high amounts of cytokines. We conclude that If-1l gene products are responsible for the low producer phenotype by efficiently interfering with NDV transcription, leading to strongly reduced intracellular levels of cytokine inducing viral dsRNA intermediates.

Inhibition by interleukin-4 of constitutive beta interferon synthesis in mouse macrophages.

We have found that interleukin-4 (IL-4) abrogated constitutive beta interferon (IFN-beta) synthesis in mouse macrophages. Analysis of endogenous IFN mRNA by reverse transcription-PCR clearly documented the presence of IFN-beta but not of IFN-alpha transcripts in RNA from bone marrow-derived macrophages (BMM). Culture of bone marrow cells for 7 days in the presence of macrophage colony-stimulating factor plus IL-4 or treatment of mature BMM with IL-4 for 3 to 4 days resulted in pronounced inhibition of IFN-beta transcript levels. As a consequence, BMM became highly permissive to viral infection and induction of the IFN-responsive 2',5'-oligoadenylate synthetase was inhibited. In view of the evidence for low-level constitutive secretion of IFN in vivo, it is possible that IL-4 influences the host's antiviral defense system.