Catherine Y. Lau

Turning down the heat: new routes to inhibition of inflammatory signaling by prostaglandin H2 synthases

Many nonsteroidal anti-inflammatory drugs act by inhibiting the cyclooxygenase activity of prostaglandin H2 synthase (PGHS), a key enzyme in the biosynthesis of prostaglandins. Gastric toxicity remains a serious problem with the current drugs, however. Recent advances in the understanding of PGHS now suggest two possible approaches to producing drugs with fewer side effects.

Cytokine-modulating activity of tepoxalin, a new potential antirheumatic.

Tepoxalin is a new dual cyclooxygenase/5-lipoxygenase anti-inflammatory compound currently under clinical investigation. It has been shown to possess anti-inflammatory activity in a variety of animal models and more recently to inhibit IL-2 induced signal transduction. The current study was conducted to evaluate the cytokine modulating activity of tepoxalin and the role of iron in these effects. In human peripheral blood mononuclear cells (PBMC) stimulated with OKT3/PMA, tepoxalin inhibited lymphocyte proliferation with an IC50 of 6 microM. Additionally, it inhibited the production of LTB4 (IC50 = 0.5 microM) and the cytokines IL-2, IL-6 and TNF alpha (IC50 = 10-12 microM). Cytotoxicity was not demonstrated at these concentrations. Add-back experiments with either cytokines (IL-2 or IL-6), LTB4 or conditioned media failed to restore the proliferative response in the presence of tepoxalin. However, the concurrent addition of iron (in the form of ferrous or ferric chloride and other iron salts) reversed the inhibition of proliferation caused by tepoxalin. Tepoxalin also inhibits the activation of NF kappa B, a transcription factor which acts on several cytokine genes. Tepoxalins effect on NF kappa B is also reversed by the addition of iron salts. These data suggest that the action of tepoxalin to inhibit proliferation in PBMC may be at least in part due to its ability to reduce the amount of available iron resulting in decreased activation of NF kappa B and subsequent inhibition of cytokine production.

A mutant human T-cell line producing immunosuppressive factor(s)

6T-CEM-20, a subclone of a 6-thioguanine-resistant mutant derived from the human-T-cell line CEM, secreted into the medium, a high titered immunosuppressive factor specific for T cells. The cell-free supernatant was very potent in suppressing, via a noncytotoxic mechanism, mitogen-activated T-cell proliferation, cytotoxic T-cell functions, and pokeweed mitogen (PWM)-stimulated plaque-forming cells (PFC). Lower dilutions of the supernatant (10(-1)-10(-2] abrogated T-cell functions within 24 hr whereas higher dilutions (10(-3)-10(-7] required a culture period of up to 4 days with lymphocytes to arrest T-cell activities. The suppressive activity was most pronounced when the factor was added in the early part of the culture period. The factor was sensitive to heat treatment and both low and high pH (most stable at physiological pH). Preliminary purification with column chromatography indicates that the active moiety was contained in the high-molecular-weight fraction (MW greater than 200,000). Data from coculture experiments suggested that T lymphocytes, which were exposed for 5-12 hr to the active supernatant or the partially purified material, suppressed allogeneic T-cell proliferation in a dose-related manner. This suppressor factor which we called suppressor-activating factor (SAF) might have activated a suppressor population or induced the production of a suppressor factor which in turn mediated the observed suppression. Both the molecular structure and the detailed mechanism of action are under investigation.

Purification, characterization, and structural elucidation of the active moiety of the previously called "suppressor activating factor (SAF)".

Upon extensive purification of the serum-free supernatant produced by a mutant T cell line (6T-CEM), an immunosuppressive activity was found to reside in an oxidized product of spermine, spermine dialdehyde (SDA). The activity was purified to homogeneity from a serum-free supernatant by using gel filtration chromatography and reverse-phase C18 HPLC. Fast Atom Bombardment (FAB) mass spectral analysis revealed its MW to be 202 and Electron Impact (EI) analysis of the acetylated material identified the purified molecule to be spermine. In the presence of human or rodent plasma, spermine exhibited no immunosuppressive activity up to 2 mg/ml. However, when assayed in the presence of FCS, which contains polyamine oxidase (PAO), spermine is oxidized to its corresponding dialdehyde which is active at 0.1 microM/ml. We have previously described a high molecular weight suppressor activating factor (SAF) found in the serum-containing supernatant of the 6T-CEM cell line. Our preliminary biological data suggest that SDA is probably responsible for the immunosuppressive activities previously observed for the SAF. The strong affinity of SDA for proteins and thiocompounds may account for the apparent high MW previously reported for SAF.

Tepoxalin, a novel immunomodulatory compound, synergizes with CsA in suppression of graft-versus-host reaction and allogeneic skin graft rejection.

Tepoxalin, a dual 5-lipoxygenase and cyclooxygenase inhibitor with nonsteroidal antiinflammatory effects, has recently been shown to suppress NFkB transactivation and inhibit T cell proliferation via a mechanism very different from cyclosporine (CsA). In this report, we demonstrate that this novel immuno-suppressive effect of tepoxalin is manifested in in vivo transplantation models. Tepoxalin suppressed murine spleen cell proliferation in a mixed lymphocyte reaction (MLR) with an IC50 of 1.3 μM. Coadministration of tepoxalin and CsA in MLR cultures showed an additive inhibitory effect. Oral administration of tepoxalin at 12 mg/kg/day to mice suppressed local graft-versus-host (GVH) responses by about 40% (n=10). Combination of tepoxalin and CsA at suboptimal doses syner-gized their immunosuppressive effects on GVH responses (n=20). In skin transplantation, the median survival time of allogeneic BALB/cByJ (H-2d) mouse skin grafted onto C3H/HeJ (H-2k) mice was 10.5 days (n=8), and was prolonged to 15.0 days (n=9) for recipient mice administered tepoxalin at 50 mg/kg/day. Co-administration of suboptimal doses of tepoxalin (12.5 mg/kg/day) and CsA (50 mg/kg/day) prolonged skin graft rejections dramatically (55% of the grafts survived for more than 40 days, n=9). Taken together, these results demonstrate that tepoxalin is a potent immunomodulatory compound that, when combined with CsA, provides synergistic immunosuppressive activity. The fact that tepoxalin and CsA act on different transcription factors, NFkB and NFAT respectively, might explain the synergistic suppressive effects when both compounds were used. Tepoxalin could be an important addition to the cohort of immunosuppressive therapies currently used in solid organ and bone marrow transplantations.

Intestinal T Cells in CD8α Knockout Mice and T Cell Receptor Transgenic Mice

T cells distributed at the epithelium of the intestines are called intraepithelial lymphocytes (IEL). IEL are heterogenous populations comprising of αβ and γδ T cells. The majority of IEL express CD8 mainly as homodimers of the a subunit (CD8αα), whereas T cells in other peripheral lymphoid organs express CD8 as heterodimers of the α and β subunits (CD8αβ) (reviewed in Ref. 3). Autoreactive T cells have been reported to be present in IEL population4 and therefore IEL are suggested to mature through an extra- thymic pathway. In the CD8α knockout mice, the CD8 co-receptor was shown to be required for thymic ontogeny of cytotoxic T cells but not helper T cells.1 In the H-Y transgenic mice, CD8+ T cells with the male H-Y antigen specific T cell receptor are thymically deleted in the male but selected in the female mice.2 In this report, the criteria for the ontogeny and function of IEL were studied in the CD8α knockout mice and in the H-Y T cell receptor transgenic mice. In these transgenic and knockout models, the antigen specificity of IEL can be defined by the H-Y specific T cell receptor and the defects observed for IEL in CD8α knockout mice would be a reflection of the indispensable role of CD8α in the ontogeny and function of IEL.

Use of quantitative polymerase chain reaction to study cellular retinoic acid-binding protein-II mRNA expression in human skin.

Publisher Summary The use of linked reverse transcription–polymerase chain reactions (RT–PCR) to quantitate specific mRNAs is rapidly gaining favor over Northern blot or RNase protection analyses due to its convenience, speed, and sensitivity. However, accurate quantitative results require careful design of proper controls. The system described in this chapter uses a synthetic RNA generated by in vitro transcription from the cDNA of human CRABP-II, with a small deletion in the coding region. Using identical primers, the test sample and internal control amplify competitively in each reaction tube. This strategy avoids problems due to tube-to-tube variations, unequal amplification efficiencies, and the plateau effect that arise with external controls or unrelated internal controls. A titration system is used to quantitate CRABP-II mRNA accurately. A serial dilution of the sample RNA, together with a fixed amount of internal control RNA, is co-reverse transcribed by a CRABP-II-specific primer. An aliquot of each RT reaction is subsequently amplified by PCR. Because the target and control cDNAs amplify with identical efficiencies, the ratio of the two products is cycle number independent, and the products can be allowed to accumulate to high levels. Following polyacrylamide gel electrophoresis (PAGE), the two products are visualized by ethidium bromide staining.