Potent inhibition of cytokine production from intestinal lamina propria T cells by phosphodiesterase-4 inhibitory thalidomide analogues.

In Crohn's disease, intestinal lamina propria (LP) T cells overproduce TNF-alpha and IFN-gamma, and clinical and animal studies indicate that this is pathogenic. Thalidomide influences cytokine production by leukocytes, inhibiting macrophage TNF-alpha, and is beneficial in treating Crohn's disease. Chemical analogues have been synthesized that may lack teratogenic and other side effects of thalidomide. We tested three analogues [selective cytokine inhibitory drugs (SelCIDs) A, B, and C, all potent PDE4 inhibitors] for effect on TNF-alpha, IFN-gamma, and IL-10 production by and on proliferation of intestinal LP mononuclear cells after T-cell stimulation and results were compared with those for peripheral blood leukocytes (PBL). While thalidomide itself had little effect, the SelCIDs were potent inhibitors, with relative inhibitory potencies: A> or =B>>C. The LP T cells were less sensitive to inhibition by the SelCIDs than were PBL. Since highly pre-activated PBL were even less sensitive, activation state alone can account for the responsiveness of intestinal LP T cells. Thalidomide analogues could play a role in treating Crohn's disease and other inflammatory disorders.

A soluble factor produced by lamina propria mononuclear cells is required for TNF-alpha enhancement of IFN-gamma production by T cells.

The role of TNF-alpha in the mucosal inflammation of Crohn's disease has been demonstrated by the prolonged clinical responses and/or remissions among patients receiving i.v. infusion of anti-TNF-alpha. A correlation between TNF-alpha and elevated IFN-gamma production is suggested by the reduction in the number of IFN-gamma producing lamina propria mononuclear cells (LPMC) found in colonic biopsies from anti-TNF-alpha-treated patients. The aim of this study was to define the mechanism of TNF-alpha-augmented mucosal T cell IFN-gamma production. In this paper we present evidence that cultured LPMC secrete a factor which acts on preactivated T cells in concert with TNF-alpha to augment IFN-gamma production. This activity is independent of IL-12 and IL-18, the well-documented potentiators of IFN-gamma expression, and is not produced by PBMC. Peripheral blood PHA-activated T cells incubated in supernatants from LPMC became responsive to TNF-alpha by increasing IFN-gamma output upon stimulation. These results are consistent with a model in which LPMC, but not PBMC, release an unidentified substance when cultured in vitro with low dose IL-2. This substance can act on preactivated peripheral T cells, as well as on lamina propria T cells, conditioning them to respond to TNF-alpha by increased IFN-gamma secretion upon stimulation. Expression of this factor in the gut mucosa could contribute to up-regulation of the Th1 response in the presence of TNF-alpha, and could be important for mucosal immunoregulation.

A greater variability in the 3' flanking region of the IL-6 gene in patients with systemic lupus erythematosus (SLE).

The causes of the aberrant constitutive expression of cytokines in SLE have not been elucidated yet, but alterations in cytokine gene structure could be a contributing factor. By RFLP (Restriction Fragment Length Polymorphism) analysis of genomic DNA, we found a higher incidence of allelic, higher MW XbaI bands in the IL-6 genes of 9/57 SLE patients vs 1/36 unrelated controls (p = 0.05) HLA DR/DQ typing of the polymorphic patients revealed they were all DQ beta 6. The study of one family indicated that the XbaI polymorphic patient and her polymorphic unaffected offspring had higher than normal levels of constitutive IL-6 mRNA. The SLE-associated IL-6 XbaI restriction alleles had duplications of AT-repeat sequences, approximately 500 bp downstream of the 2nd polyadenylation site, in an AT-rich mini-satellite with similarities to Matrix Associated Regions (MARs), that may be important in DNA replication and in gene expression. These are novel observations that suggest that, in SLE, there is increased variability in the 3' flanking region of the IL-6 gene. This variability may be related to the aberrant IL-6 expression that was reported by us and others in this disease.

Potentiation of lipopolysaccharide-induced tumor necrosis factor-alpha expression by 1,25-dihydroxyvitamin D3.

The immunomodulatory hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to suppress T-cell proliferation and interleukin-2 synthesis as well as B-cell immunoglobulin synthesis, while stimulating many macrophage functions. We have previously shown increased synthesis of interleukin-1 beta in lipopolysaccharide (LPS)-stimulated U937 cells after pretreatment with 10 nmol/L 1,25-(OH)2D3. We now show that 1,25-(OH)2D3 also primes the increase in U937 cell tumor necrosis factor (TNF)-alpha-accumulated mRNA after activation with LPS; 50% effective concentration (EC50) for the LPS-induced expression of TNF-alpha mRNA was decreased by two orders of magnitude after incubation with 10 nmol/L 1,25-(OH)2D3. Pretreatment of U937 cells with 10 nmol/L 1,25-(OH)2D3 also increased subsequent LPS-induced TNF-alpha mRNA expression by twofold and cell-associated TNF protein levels by more than ninefold. This potentiation was steroid-specific for 1,25-(OH)2D3 because dexamethasone inhibited TNF-alpha mRNA. The potentiation required prior exposure to 1,25-(OH)2D3 for more than 6 hours and was clearly seen after 12 hours. The finding that the sensitivity of the U937 cell monokine response to LPS was dramatically increased by 1,25-(OH)2D3 and the delayed effect on the LPS-stimulated TNF-alpha gene transcript levels indicated that 1,25-(OH)2D3 may be altering the expression of a protein(s) in the U937 cell LPS-signal transduction pathway. In fact, 1,25-(OH)2D3 induced expression of the mRNA for CD14, the high affinity, cell-surface glycoprotein receptor for LPS, which could account for the enhancement of LPS-stimulated monokine gene expression by 1,25-(OH)2D3. Thus, local monokine gene expression may be regulated by both the amount and the temporal entry of the vitamin D hormone and activator(s) into the inflammatory microenvironment.

Stimulus-specific 1,25(OH)2D3 modulation of TNF and IL-1-beta gene expression in human peripheral blood mononuclear cells and monocytoid cell lines.

It is known that 1,25(OH)2D3 inhibits IL-2, IFN-gamma, and GM-CSF mRNA accumulation in activated T cells. While 1,25(OH)2D3 enhances macrophage competence, its effect on cytokine gene expression in monocytes is less well defined. Using Northern blot analysis, we examined the effect of 1,25(OH)2D3 pretreatment on IL-1 beta and TNF gene expression in LPS- and PHA-stimulated human PBMNC and several human myeloid cell lines (U937 and THP1). In PBMNC, preincubation had no effect on the steady-state level of LPS-induced TNF or IL-1 beta mRNA. When PHA was used to stimulate pretreated PBMNC, a 60-80% inhibition of TNF mRNA levels was observed. There was no effect on IL-1 beta mRNA. In U937 cells, 1,25(OH)2D3 preincubation resulted in a 4-fold increase in the level of TNF and IL-1 beta mRNA levels. Pretreatment had no effect on TNF or IL-1 beta gene expression in THP1 cells. The observation that PHA-induced TNF gene expression is modulated by 1,25(OH)2D3 is novel. Possible mechanisms by which 1,25(OH)2D3 preincubation may influence mitogen-specific inducible cytokine gene expression in different cell types are discussed.

The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o

Facial vein of the rabbit. Intracellularly recorded hyperpolarization of smooth muscle cells induced by beta-adrenergic receptor stimulation.

A segment of the facial vein of the rabbit gains myogenic tone when placed under tension in a tissue bath (Winquist and Bevan, 1977); low frequency electrical stimulation of intramural nerves, or application of norepinephrine or isoproterenol produces relaxation, which is blocked by propranolol (1.0 microM). We measured membrane potential of single smooth muscle cells with microelectrodes during this beta-adrenergic receptor-mediated relaxation of the vein segment. Hyperpolarization (up to 30 mV) invariably accompanied relaxation. Average maximum resting potential recorded in normal Krebs solution was -46.8 +/- 3.6 mV (mean +/- SE); this increased to -70.8 +/- 3.0 mV when high concentrations (greater than 10 nM) of isoproterenol were added to the bath. Tone and membrane potential were very sensitive to intramural nerve stimulation. At 3-4 Hz, relaxation was about half maximal and hyperpolarization was 6-9 mV. The maximum hyperpolarization to nerve stimulation was 20-30 mV, about the same magnitude as that produced by isoproterenol. Hyperpolarization clearly preceded relaxation by about 0.2 second. The magnitude of hyperpolarization in facial vein is much larger (2-10 times or more) than that recorded in other tissues during inhibition produced by stimulation of beta-adrenergic receptors. The large hyperpolarization may augment the effectiveness of inhibition (by curtailing potential dependent influx of Ca++o, for example) in this tissue compared to that in other tissues in which hyperpolarization is slight or absent. The ionic mechanism underlying hyperpolarization resulting from stimulation of beta-adrenergic receptors remains unclear.