Cleaved secretory leucocyte protease inhibitor as a biomarker of chymase activity in allergic airway disease.

BACKGROUND: Secretory leucocyte protease inhibitor (SLPI), which is present in many physiological fluids including saliva, sputum and nasal discharge, is the most effective inhibitor of chymase. Previously, we demonstrated that chymase is able to cleave SLPI and that the cleaved portion, cSLPI, is a biomarker of chymase activity. OBJECTIVE: We investigated the potential of cSLPI as a biomarker of chymase activity in subjects with allergic rhinitis (AR) and asthmatic airway disease. METHODS: Baseline sputum samples were collected from atopic asthmatics and healthy controls (HC). Nasal lavages (NAL) were performed in subjects with AR both at baseline and following a nasal challenge with allergen or placebo. Levels of cSLPI and chymase were determined by Western analysis, and tryptase and alpha-2 macroglobulin were measured by immunoassay. RESULTS: As compared with HC, asthmatics showed a significant increase in baseline cSLPI/total SLPI ratios and an increase in chymase levels. There was a high correlation of cSLPI/SLPI ratios to chymase levels in normal individuals and untreated asthmatics. In the NAL of patients with AR, as compared with placebo, allergen challenge increased inflammatory biomarkers, including cSLPI/SLPI ratios, chymase levels, tryptase levels and alpha2-macroglobulin levels. Correlations were observed between cSLPI/SLPI ratios and chymase levels and cSLPI/SLPI ratios and alpha2-macroglobulin levels; no correlation was seen between cSLPI/SLPI ratios and tryptase levels. CONCLUSION: Our data indicate that cSLPI reflects chymase activity in AR and asthma. Hence, cSLPI may serve as a biomarker for disease activity and for monitoring the efficacy of novel anti-inflammatory treatments in chymase-mediated diseases.

Requirement of PI3-kinase activity for the nuclear transport of prolactin in cloned murine T lymphocytes.

The murine T-cell clone, L2, requires both IL2 and PRL to proliferate. Proliferation and selected IL2-driven gene expression are blocked by treatment with rapamycin. Since prolactin translocation to the nucleus is IL2 dependent and required for proliferation, experiments were performed to identify activation pathways that might be involved in nuclear transport and proliferation. L2 cells were stimulated with IL2 in the presence and absence of the mTOR inhibitor rapamycin, PI3-kinase inhibitors (wortmannin, LY294002), the p38 MAP kinase inhibitor SB203580 and the vitamin D analog calcipotriol. The immunosuppressant rapamycin markedly inhibited IL2-induced proliferation and prolactin translocation to the nucleus. Similarly, wortmannin and LY294002 inhibited IL2-induced proliferation and markedly decreased the amount of prolactin transported to the nucleus. SB203580 and calcipotriol partially inhibited IL2-induced proliferation but had no effect on prolactin translocation. None of the inhibitors affected Lucifer Yellow uptake indicating that rapamycin, wortmannin and LY294002 did not inhibit early endosomal formation but rather worked to inhibit prolactin translocation at a later point in the retrograde transport pathway.

Characterization of kappa-opioid receptor transcripts expressed by T cells and macrophages.

We have found that the immature T cell lines R1.1 and DPK and the macrophage lines P388D1 and WEHI-3 also express kappa-opioid receptor (KOR) mRNA. Characterization of the KOR transcripts in both brain tissue and these T cells has revealed both the normal full-length as well as a truncated form of the mRNA. Our results show that the truncated transcript lacks the second exon. Primary macrophages express this truncated form of the transcript in the absence of detectable levels of the full-length form. These results suggest a degree of heterogeneity in the expression of the opioid receptors which has not previously been reported.

Isolation and analysis of a T cell clone variant exhibiting constitutively phosphorylated Ser133 cAMP response element-binding protein.

In driving T cell proliferation, IL-2 stimulates a new program of gene expression that includes proliferating cell nuclear antigen (PCNA), a requisite processivity factor for DNA polymerase delta. PCNA transcription is regulated in part through tandem CRE sequences in the promoter and CRE binding proteins; IL-2 stimulates CREB phosphorylation in the resting cloned T lymphocyte, L2. After culturing L2 cells for greater than 91 days, we consistently isolate a stable variant that exhibits constitutive CREB phosphorylation. L2 and L2 variant cells were tested for IL-2 responsiveness and rapamycin sensitivity with respect to specific kinase activity, PCNA expression and proliferation. In L2 cells, IL-2 stimulated and rapamycin inhibited the following: cAMP-independent CREB kinase activity, PCNA expression and proliferation. In L2 variant cells, CREB kinase activity was constitutively high; IL-2 stimulated and rapamycin blocked PCNA expression and proliferation. These results indicate that IL-2 induces a rapamycin-sensitive, cAMP-independent CREB kinase activity in L2 cells. However, phosphorylation of CREB alone is not sufficient to drive PCNA expression and L2 cell proliferation in the absence of IL-2.

Lymphocyte-specific inducible expression of potassium channel beta subunits.

Many studies have shown that voltage-gated potassium (Kv) channel activity is essential for T-lymphocyte proliferation. The IL-2-inducible neuroimmune gene, I2rf5 is the mouse homologue of the rat Kv beta 2 subunit. In this study we show that in addition to constitutive expression in adult murine brain, expression of Kv channel subunits beta 1.1 and beta 2.1 is inducible in a cloned T-helper cell line stimulated with IL-2 and in normal murine splenocytes stimulated with Con A or LPS. This expression pattern appears to be lymphocyte specific, because stimulated fibroblasts and vascular smooth muscle cells do not express Kv beta channel subunit mRNA. These observations suggest that Kv beta subunit expression is tissue specific and inducible in stimulated lymphocytes. Because Kv beta subunits modulate K+ channel activity, the inducible and variable expression of these subunits in lymphocytes may represent an additional regulatory mechanism for lymphocyte proliferation.

Sequence of kappa-opioid receptor cDNA in the R1.1 thymoma cell line.

To our knowledge, this is the first demonstration of kappa-opioid receptor mRNA in cells of the immune system. While the presence of opioid receptors on cells of the immune system has been controversial, cell-binding analysis has indicated that the kappa-opioid receptor is expressed by the immature T cell line R1.1. We have developed a reverse transcriptase-polymerase chain reaction protocol to amplify the mRNA extracted from R1.1 cells with primers derived from the cDNA sequence of the mouse kappa-opioid receptor. Nucleotide sequences of the amplified products were examined and two populations of cDNA were detected which differ in the 5' region upstream of the ATG start codon. Comparison of these sequences to the previously published kappa-opioid receptor cDNA sequence suggests the presence of an intron-exon junction in the 5' non-coding region.

Inhibition of interleukin-1 and tumor necrosis factor-alpha synthesis following treatment of macrophages with the kappa opioid agonist U50, 488H.

Previous reports from this laboratory, and others, have shown that exogenous mu and kappa opioids modulate both cellular and humoral immune responses. Our earlier work has suggested that accessory cells may serve as a target for the direct effects of kappa opioid compounds. In the present study, the function of the macrophage cell line P388D1 was modulated by the kappa-selective opioid agoinst U50,488H (trans-3,4-dichloro-N-methyl-N-[7-(1- pyrrolidinyl)cyclohexyl]benzene-acetamide methanesulfonate). Lipopolysaccharide-induced interleukin (IL)-1 and tumor necrosis factor-alpha production were inhibited after the administration of nanomolar concentrations of U50,488H. Furthermore, inhibition of IL-1 produced by the P388D1 cell line was reversed by both the classical opioid antagonist naloxone and by the kappa opioid receptor antagonist norbinaltorphimine. Examination of IL-1 mRNA levels in P388D1 by northern blot analysis showed that the inhibition mediated by U50, 488H apparently occurred at the level of transcription. On the other hand, U50,488H failed to modulate the production of IL-6 by this macrophage-like cell line. In addition, U50,488H failed to modulate the production of either IL-1 or tumor necrosis factor-alpha from the macrophage-like cell line RAW 264.7, an indication that subpopulations of macrophages exist with different sensitivities to opioids. These results are consistent with a growing body of data which suggests that a component of the inhibition mediated by opioid compounds involves a reduction in the production of cytokines.

Detection of kappa-opioid receptor mRNA in immature T cells.

The R1.1 cell line has been shown to express kappa-opioid receptors on the cell surface. Our analysis shows that the R1.1 cell line exhibits a CD4NEG CD8NEG CD3LOW CD25LOW cell surface phenotype, characteristic of thymocytes in one of the early stages of differentiation. We have developed reverse transcriptase polymerase chain reaction (RT-PCR) conditions that permit the detection of mRNA coding for the kappa-receptor. Using cell fractionation techniques we have isolated CD4NEG CD8NEG thymocytes, and analysis by RT-PCR shows that these primary immature thymocytes also express the kappa-opioid receptor. We hypothesize that the expression of kappa-opioid receptor may be a marker which is characteristic of immature T development.