Inhibition of LFA-1 Mediated T-Cell Motility by Naphthoquinones

• Abstract
• Materials and Methods
• References

Abstract

An in vitro T-cell migration assay has been established that can be used to study the effects of compounds on the development of T-cell polarisation with HuT-78 T lymphocytes. This assay indicates the ability of compounds tested to inhibit the inflammatory response by decreasing LFA-1-mediated T-cell motility. The effect of a series of naturally occurring quinone isolates on motility has been evaluated in this assay. Distinct differences have been observed between naphthoquinones, dihydrofuranonaphthoquinones and anthraquinones

The migration of T lymphocytes from the blood stream into surrounding tissues and lymph nodes is critical to ensure that an effective immune response is generated. This process also contributes to the mechanism of inflammation in the body. Migration of the cells toward antigenic challenge is permitted by cytoskeletal-based rearrangements, generated in response to integrin activation on the surface of the T lymphocyte. One of the best characterised integrin adhesion molecules on T lymphocytes is lymphocyte functional antigen-1 (LFA-1) [1], [2].

Activation of LFA-1 on T cells by stimulatory monoclonal antibodies to the adhesion molecule (anti-LFA-1) induces the same migratory phenotype as that acquired upon activation of LFA-1 by its cognate physiological ligand (ICAM-1). This in vitro induced migratory phenotype mimics the ability of lymphocytes to migrate to sites of inflammation in the body, as the interaction between LFA-1 and its physiological ligand (ICAM-1) are crucial for cell migration and inflammation in vivo. Weitz-Schmidt and co-workers [3] demonstrated that the anti-inflammatory-immunosuppressive properties exhibited by statins (cholesterol-lowering drugs) are mediated by their binding to and inhibition of the LFA-1 adhesion molecule.

As part of an ongoing study into the use of natural products as anti-inflammatory agents, the effects of a series of quinones on T cell motility has been investigated. Quinones are an important group of pharmacologically active pigments widely distributed in nature. They demonstrate a range of physiological effects including antimicrobial, anti-inflammatory and anticancer activity [4]. The mechanism of action of naphthoquinones has received much attention and is still under investigation. To date the most important biochemical reaction of quinones is their reversible reduction to the corresponding hydroquinone [5]. The theory of free radical generation may be consistent as a mechanism for cytotoxicity and enzyme inhibition; however, it does not fully explain the observed anti-inflammatory activity of many quinones.[]

A range of quinones 1 – 7 isolated from a series of plant cell cultures currently under investigation in our laboratories [6], [7] have been evaluated in the LFA-1 mediated T-cell motility assay. Three structural classes of quinones were investigated in this study including naphthoquinones (1, 2), dihydro-furanonaphthoquinones (3 – 6) and an anthraquinone (7). All compounds were tested at 10 μM initially ([Fig. 1]) and those compounds (1 and 2) that substantially inhibited motility were then tested in a dose-response study ([Fig. 2]). The PKC inhibitor Go6976 (1 μM) was used as a positive control for migration inhibition [1]. Untreated cells were used as a control of the extent of deformation; LFA-1 in the absence of any test compound induced motility (deformation index > 3) in every cell observed. While all compounds (10 μM) significantly (P < 0.05) reduced motility compared to untreated controls, a relatively high dose was employed to achieve a minimal effect. Compared to the positive control Go6976 (1 μM), the only test compounds (at 10 μM) to substantially inhibit motility were the structurally related naphthoquinones 7-methyljuglone (1) and plumbagin (2) where cell motility dropped to 7.7 ± 3.8 % and 2.3 ± 1.6 %, respectively. A subsequent dose-response study showed a concentration dependent relationship ([Fig. 2]). The percentage of motile cells ranged from 13.3 ± 2.9 % and 21.6 ± 2.1 % for (1) and (2), respectively, at 100 nM, down to 0.8 ± 0.3 % and 0.6 ± 0.1 % at 30 μM. Of the furanonaphthoquinones, 7-hydroxydunnione (4) decreased cell motility markedly but not significantly. Apoptotic bodies were also identified in cells incubated with this compound ([Fig. 3]). The anthraquinone (7) had no effect on motility.

A cytotoxicity study using a range of concentrations was carried out to investigate the effect on lactate dehyrogenase (LDH) release ([Fig. 4]). Negligible toxicity was observed below 100 μM for tested compounds. At 100 μM and 300 μM, toxicity increased to 16.6 ± 17.1 % and 55.1 ± 3.0 % for plumbagin (2). At the same concentrations, toxicity from 7-methyljuglone (1) remained low at 5.0 ± 2.9 % and 7.6 ± 3.0 %, respectively [6]. The inhibitory effect of 1 and 2 on LFA-1-mediated polarity of HuT 78 cells is dose-dependent and is indicative of an inhibitory effect by naphthoquinones on elements involved in the process of T-cell chemoattraction and movement towards areas of inflammation. The inhibition in the number of motile cells caused by either the furanonaphthoquinones or the anthraquinone is much less pronounced and may indicate a non-specific activity. The inhibitory effects observed with 7-methyljuglone (1) and plumbagin (2) are not mediated by or associated with cell toxicity, which is only evidenced at a concentration of 100 μM. While the mechanism of action is unclear, further investigation might focus on the potential inhibition of 12(S)-HETE and the deactivation of protein kinase C, which has been reported for some naphthoquinones [8].

Materials and Methods

The naphthoquinones 7-methyljuglone (1) and plumbagin (2) were obtained from suspension cultures of Dionaea muscipula and Drosera species [7]. Dunnione (3) and 7-hydroxydunnione (4) were isolated from Streptocarpus × hybridus var Ruby. α Dunnione (5), 7-hydroxy-α-dunnione (6) and 1-hydroxy-2-methylanthraquinone (7) were isolated from Streptocarpus dunnii [9]. Isolates were characterised by their physical and spectroscopic properties.

Cell culture: HuT 78 T lymphocytes (LGC Promochem) were cultured in RPMI 1640 (Gibco) supplemented with 1 % penicillin/streptomycin, 1 % L-glutamine, 40 mL FBS, 5 mL HEPES at 10 μM, 5 mL glucose at 4.5 g/L, 5 mL sodium pyruvate at 1 μM and 2 mL sodium bicarbonate at 1.5 g/L in a humidified atmosphere at 37 °C and 5 % CO₂. Cells were seeded at a density of 0.5 × 10⁶ cells per mL in T-75 cm² flasks with filter lid and maintained at a density of 0.5 – 0.9 × 10⁶ cells per mL. Cells were subcultured every 3 to 4 days.

Treatments: Go6976 at 1 μM (Calbiochem), a selective PKC-α and -β inhibitor [10], was used as a positive control of motility inhibition [2]. Stock concentrations of test compounds and Go6976 in DMSO were added to the cells at the appropriate volume to give the desired final concentration; the final concentration of DMSO being 0.1 %. The cell/drug mixture was incubated for 30 minutes in a humidified atmosphere at 37 °C and 5 % CO₂. Treatments were prepared on the day of experiment.

LFA-1 bioassay: A 24-well sterile flat-bottomed tissue culture plate was coated with 250 μL per well of a 1/100 dilution of rabbit anti-mouse IgG in sterile PBS. The plate was sealed with Parafilm® and stored at 4 °C overnight. The antibody coated plate was then washed twice with sterile PBS and subsequently coated with 250 μL per well of a 1/100 dilution of LFA-1 in sterile PBS. The plate was covered and incubated in a humidified atmosphere at 37 °C and 5 % CO₂ for at least 1 hour. Drugs and controls were incubated on a second plate. The model used for this investigation was the HuT 78 T lymphocyte cell line. 10⁴ cells/well were required. Cell density was determined by a suspension of 50 μL of cells in 50 μL Trypan blue with 400 μL PBS and a haemocytometer was used to count the cells. Adjustments to the cell density were made as appropriate. Cells were dispensed into each well, and the plate was incubated at 37 °C and 5 % CO₂ for 10 minutes. The density was then checked under the microscope and adjusted accordingly if necessary. Cells were treated with the appropriate volumes of stock concentrations of test compounds in DMSO, to give the desired final concentration. Go6976 (1 μM) was used as a positive control of motility inhibition. There was no determination made of whether test compounds or Go6976 had any direct effect on the LFA-1 antibody. The cell/drug mixture was incubated in a humidified atmosphere at 37 °C and 5 % CO₂ for 30 minutes. The antibody plates were washed as before. The cell/drug mixture was added and incubated at 37 °C again, until sufficient cytoskeletal rearrangement in control untreated cells had been observed. Image Pro Plus (Media Cybernetics) was used to analyse the cells. Photographs (in triplicate) were taken of each well; each photograph containing between 8 and 21 cells. Motility was then determined by calculation of the deformation index. A deformation index greater than 3 was considered to indicate motility. Deformation Index = elongation index/circularity index [1], [2].

Cytotoxicity assay: Cytotoxicity was assessed using a LDH cytotoxicity assay (Roche). Briefly, 50,000 HuT 78 cells/well were incubated with the test compounds for 4 h at 37 °C and 5 % CO₂. 100 μL of supernatant were then removed, and added to a new 96-well plate. 100 μL of freshly prepared reaction mixture were added to this plate. The plate was protected from light with aluminium foil and left to stand at room temperature for 10 minutes. The absorbance at 492 nm was measured. Cytotoxicity is expressed as the amount of LDH released as percentage of LDH release by the positive control, Triton-X 100 (2 mM), after subtraction of spontaneous release from both values.

Data are expressed as mean ± S.E.M of n wells from three individual experiments conducted on different days. Statistical analysis was by means of 1-way ANOVA followed by Dunnett′s multiple comparison test as a post test, with significance taken as P < 0.05.

Copies of original spectra and biological activity data can be obtained from the corresponding author.

References

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Dr. Dr. Helen Sheridan

Trinity College Dublin

School of Pharmacy & Pharmaceutical Sciences

Panoz Institute

23 Westland Row

Dublin 2

Ireland

Fax: 353-1-608-2804

Email: hsheridn@tcd.ie

Email: nfrnkish@tcd.ie

References

• 1 Volkov Y, Long A, Kelleher D. Inside the crawling T cell: leukocyte function-associated antigen-1 cross-linking is associated with microtubule-directed translocation of protein kinase C isoenzymes beta(I) and delta.  J Immunol. 1998;  161 6487-95
  PubMedSearch in Google Scholar
• 2 Volkov Y, Long A, McGrath S, Ni Eidhin D, Kelleher D. Crucial importance of PKC-beta(I) in LFA-1-mediated locomotion of activated T cells.  Nat Immunol. 2001;  2 508-14
  CrossrefPubMedSearch in Google Scholar
• 3 Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, Bruns C. et al . Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site.  Nat Med. 2001;  7 687-92
  PubMedSearch in Google Scholar
• 4 Hsu Y L, Cho C Y, Kuo P L, Huang Y T, Lin C C. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through p53 accumulation via c-Jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and in vivo.  J Pharmacol Exp Ther. 2006;  318 484-94
  CrossrefPubMedSearch in Google Scholar
• 5 Fanning A, Volkov Y, Freeley M, Kelleher D, Long A. CD44 cross-linking induces protein kinase C-regulated migration of human T lymphocytes.  Int Immunol. 2005;  17 449-58
  CrossrefPubMedSearch in Google Scholar
• 6 O′Brien P J. Molecular mechanisms of quinone cytotoxicity.  Chem Biol Interact. 1991;  80 1-41
  PubMedSearch in Google Scholar
• 7 Hook I. Naphthoquinone contents of in vitro cultured plants and cell suspensions of Dionaea muscipula and Drosera species.  Plant Cell Tiss Org. 2001;  67 281-5
  CrossrefPubMedSearch in Google Scholar
• 8 Wube A A, Streit B, Gibbons S, Asres K, Bucar F. In vitro 12(S)-HETE inhibitory activities of naphthoquinones isolated from the root bark of Euclea racemosa ssp. schimperi.  J Ethnopharmacol. 2005;  102 191-6
  CrossrefPubMedSearch in Google Scholar
• 9 Nestor C. The production of quinones by in vitro cultures of Dionaea and Streptocarpus species. Dublin; Trinity College Dublin 2006
  Search in Google Scholar
• 10 Martiny-Baron M G, Kazanietz H, Mischak P M, Blumberg G, Kochs H, Hug D. et al . Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976.  J Biol Chem. 1993;  268 9194-7
  PubMedSearch in Google Scholar

Dr. Dr. Helen Sheridan

Trinity College Dublin

School of Pharmacy & Pharmaceutical Sciences

Panoz Institute

23 Westland Row

Dublin 2

Ireland

Fax: 353-1-608-2804

Email: hsheridn@tcd.ie

Email: nfrnkish@tcd.ie