Activated T cells exhibit increased uptake of silicon phthalocyanine Pc 4 and increased susceptibility to Pc 4-photodynamic therapy-mediated cell death.

Photodynamic therapy (PDT) is an emerging treatment for malignant and inflammatory dermal disorders. Photoirradiation of the silicon phthalocyanine (Pc) 4 photosensitizer with red light generates singlet oxygen and other reactive oxygen species to induce cell death. We previously reported that Pc 4-PDT elicited cell death in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro, and furthermore that Jurkat cells were more sensitive than A431 cells to treatment. In this study, we examined the effectiveness of Pc 4-PDT on primary human CD3(+) T cells in vitro. Fluorometric analyses of lysed T cells confirmed the dose-dependent uptake of Pc 4 in non-stimulated and stimulated T cells. Flow cytometric analyses measuring annexin V and propidium iodide (PI) demonstrated a dose-dependent increase of T cell apoptosis (6.6-59.9%) at Pc 4 doses ranging from 0-300 nM. Following T cell stimulation through the T cell receptor using a combination of anti-CD3 and anti-CD28 antibodies, activated T cells exhibited increased susceptibility to Pc 4-PDT-induced apoptosis (10.6-81.2%) as determined by Pc 4 fluorescence in each cell, in both non-stimulated and stimulated T cells, Pc 4 uptake increased with Pc 4 dose up to 300 nM as assessed by flow cytometry. The mean fluorescence intensity (MFI) of Pc 4 uptake measured in stimulated T cells was significantly increased over the uptake of resting T cells at each dose of Pc 4 tested (50, 100, 150 and 300 nM, p < 0.001 between 50 and 150 nM, n = 8). Treg uptake was diminished relative to other T cells. Cutaneous T cell lymphoma (CTCL) T cells appeared to take up somewhat more Pc 4 than normal resting T cells at 100 and 150 nm Pc 4. Confocal imaging revealed that Pc 4 localized in cytoplasmic organelles, with approximately half of the Pc 4 co-localized with mitochondria in T cells. Thus, Pc 4-PDT exerts an enhanced apoptotic effect on activated CD3(+) T cells that may be exploited in targeting T cell-mediated skin diseases, such as cutaneous T cell lymphoma (CTCL) or psoriasis.

TLR2 engagement on CD4(+) T cells enhances effector functions and protective responses to Mycobacterium tuberculosis.

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4(+) T cells and upregulate TCR-triggered IFN-γ secretion and cell proliferation in vitro. Here we examined the role of CD4(+) T-cell-expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag-specific T-cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4(+) T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1-like response was observed in the context of both polyclonal and Ag-specific TCR stimulation. To evaluate the role of T-cell TLR2 in priming of CD4(+) T cells in vivo, naive MTB Ag85B-specific TCR transgenic CD4(+) T cells (P25 TCR-Tg) were adoptively transferred into Tlr2(-/-) recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3 Cys-SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN-γ-secreting P25 TCR-Tg T cells 1 week after immunization. P25 TCR-Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4(+) T cells increases MTB Ag-specific responses and may contribute to protection against MTB infection.

Structural factors and mechanisms underlying the improved photodynamic cell killing with silicon phthalocyanine photosensitizers directed to lysosomes versus mitochondria.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Oxidative modification of cytochrome c by singlet oxygen.

Singlet oxygen ((1)O(2)) is a reactive oxygen species that may be generated in biological systems. Photodynamic therapy generates (1)O(2) by photoexcitation of sensitizers resulting in intracellular oxidative stress and induction of apoptosis. (1)O(2) oxidizes amino acid side chains of proteins and inactivates enzymes when generated in vitro. Among proteogenic amino acids, His, Tyr, Met, Cys, and Trp are known to be oxidized by (1)O(2) at physiological pH. However, there is a lack of direct evidence of oxidation of proteins by (1)O(2). Because (1)O(2) is difficult to detect in cells, identifying oxidized cellular products uniquely derived from (1)O(2) could serve as a marker of its presence. In the present study, (1)O(2) reactions with model peptides analyzed by tandem mass spectrometry provide insight into the mass of prominent adducts formed with the reactive amino acids. Analysis by MALDI-TOF and tandem mass spectrometry of peptides of cytochrome c exposed to (1)O(2) generated by photoexcitation of the phthalocyanine Pc 4 showed unique oxidation products, which might be used as markers of the presence of (1)O(2) in the mitochondrial intermembrane space. Differences in the elemental composition of the oxidized amino acid residues observed with cytochrome c and the model peptides suggest that the protein environment can affect the oxidation pathway.

Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization.

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.

Mycobacterial phosphatidylinositol mannoside 6 (PIM6) up-regulates TCR-triggered HIV-1 replication in CD4+ T cells.

Tuberculosis (TB) is the leading cause of mortality among those infected with human immunodeficiency virus (HIV-1) worldwide. HIV-1 load and heterogeneity are increased both locally and systemically in active TB. Mycobacterium tuberculosis (MTB) infection supports HIV-1 replication through dysregulation of host cytokines, chemokines, and their receptors. However the possibility that mycobacterial molecules released from MTB infected macrophages directly interact with CD4(+) T cells triggering HIV-1 replication has not been fully explored. We studied the direct effect of different MTB molecules on HIV-1 replication (R5-tropic strain Bal) in anti-CD3- stimulated CD4(+) T cells from healthy donors in an antigen presenting cell (APC)-free system. PIM6, a major glycolipid of the mycobacterial cell wall, induced significant increases in the percent of HIV-1 infected T cells and the viral production in culture supernatants. In spite of structural relatedness, none of the other three major MTB cell wall glycolipids had significant impact on HIV-1 replication in T cells. Increased levels of IFN-γ in culture supernatants from cells treated with PIM6 indicate that HIV-1 replication is likely dependent on enhanced T cell activation. In HEK293 cells transfected with TLR2, PIM6 was the strongest TLR2 agonist among the cell wall associated glycolipids tested. PIM6 increased the percentage of HIV infected cells and viral particles in the supernatant in a T-cell-based reporter cell line (JLTRg-R5) transfected with TLR1 and TLR2 but not in the cells transfected with the empty vector (which lack TLR2 expression) confirming that PIM6-induced HIV-1 replication depends at least partially on TLR2 signaling.

Psoriasis and cardiovascular risk factors: increased serum myeloperoxidase and corresponding immunocellular overexpression by Cd11b(+) CD68(+) macrophages in skin lesions.

BACKGROUND: Recent studies report independent associations between psoriasis, cardiovascular (CV) events and risk factors. Blood Myeloperoxidase (MPO) from activated myeloid cells is associated with CV risk mainly through lipid oxidation, induction of endothelial dysfunction and release of IL-12 from macrophages. OBJECTIVES: To elucidate associations between psoriasis and conventional CV risk factors. METHODS: We performed a cross-sectional study of 100 psoriasis patients and 53 controls, group matched on age, gender and body mass index, to assess levels of MPO in serum, as well as immunohistochemical staining from psoriasis skin lesions, psoriasis uninvolved skin, and normal skin. RESULTS: Although the groups did not differ on waist circumference, glucose, cholesterol, triglycerides, creatinine or personal history of CV events, psoriasis patients had significantly higher waist-to-hip ratios, blood pressures, proportion of current smokers, and lower high density lipoprotein level than controls. Serum MPO level was elevated 2.5 fold (P<0.001) in psoriasis patients, even after adjusting for the CV risk factors on which the groups differed. MPO did correlate with coronary artery calcification, carotid plaque, carotid intima media thickness and flow mediated dilation, but did not correlate with psoriasis severity. However, MPO was highly expressed in lesional psoriatic skin and colocalized predominantly with CD45(+) CD11b(+) leukocytes. CD11b(+) cell density correlated with circulation MPO levels. CONCLUSION: Lesional skin CD11b(+) leukocytes activated to generate MPO may contribute to serum levels of MPO. Lesional CD11b(+) cell activity may be an alternative measure of disease burden to PASI that underlies the MPO biomarker for systemic inflammation related to Cardiovascular Disease.

Photo-oxidation of cardiolipin and cytochrome c with bilayer-embedded Pc 4.

Singlet oxygen, (1)O(2), is produced by absorption of red light by the phthalocyanine dye Pc 4, followed by energy transfer to dissolved triplet molecular oxygen, (3)O(2). In tissues, Pc 4 concentrates in lipid bilayers, and particularly in mitochondrial membranes, because of its positive charge. Illumination of cells and tissues with red light after uptake of Pc 4 results in cell death. The potential initial chemical steps that result in cellular dysfunction have been characterized in this study. Both unsaturated acyl chains of phospholipids and proteins are identified as targets of oxidation. Tetra-linoleoyl cardiolipin was oxidized in both liposomes and mitochondria after Pc 4-mediated (1)O(2) generation. Evidence for the formation of both mono- and bis-hydroperoxide adducts of single linoleoyl side chains is provided by ESI-MS and ESI-MS/MS. Similarly, illumination of Pc 4 in liposomes and mitochondria resulted in cytochrome c oxidation as detected by oxidation of His 26 in the peptide H(26)*KTGPNLHGLFGK, further supporting the potential use of this peptide as a biomarker for the presence of mitochondrial oxidative stress characteristic of (1)O(2) in vivo (J. Kim et al., Free Radic. Biol. Med. 44:1700-1711; 2008). These observations provide evidence that formation of lipid hydroperoxides and/or protein oxidation can be the initial chemical steps in Pc 4-mediated induction of apoptosis in photodynamic therapy.

Photophysics of zinc (II) phthalocyanine polymer and gel formulation.

The photophysical properties of lipophilic phthalocyanines encapsulated into a polymer and two different gels were studied in order to predict their photosensitizing efficacy in vivo. Photophysical techniques for solid phase were adapted for light dispersing samples. Gel formulation of two tetrasubstituted phthalocyanines, tetra-t-butylphthalocyaninato zinc(II) (1), tetrakis(1,1-dimethyl-2-phthalimido)ethylphtalocyaninatozinc(II) (2) and two octasubstituted phthalocyanines, 2,3,9,10,16,17,23,24-octakis(decyloxy)phthalocyaninatozinc(II) (3) and 2,3,9,10,16,17,23,24-octakis[(N,N-dimethylamino)ethylsulfanyl]phthalocyaninatozinc(II) (4) were investigated for their possible use in photodynamic therapy for topical purposes. Supporting the fact that gel formulation improves the photophysical properties of phthalocyanines, singlet molecular oxygen quantum yield (Phi(Delta)) values for 1-4 zinc(II) phthalocyaninates in Lutrol F 127-Cremophor RH 40 were 0.60, 0.60, 0.20 and 0.26, respectively. Permeation studies showed that no release of phthalocyanines occurs, thus indicating there should be no risk of generalized skin photosensitivity in areas other than the dye-deposition site.

Delivery of the photosensitizer Pc 4 in PEG-PCL micelles for in vitro PDT studies.

The silicon phthalocyanine Pc 4 is a second-generation photosensitizer that has several properties superior to other photosensitizers currently approved by the FDA, and it has shown significant promise for photodynamic therapy (PDT) in several cancer cells in vitro and model tumor systems in vivo. However, because of the high hydrophobicity of Pc 4, its formulation for in vivo delivery and favorable biodistribution become challenging. To this end, we are studying encapsulation and delivery of Pc 4 in block copolymer micelles. Here, we report the development of biocompatible PEG-PCL micelle nanoparticles, encapsulation of Pc 4 within the micelle core by hydrophobic association with the PCL block, and in vitro PDT studies of the micelle-formulated Pc 4 in MCF-7c3 human breast cancer cells. Our studies demonstrate efficient encapsulation of Pc 4 in the micelles, intracellular uptake of the micelle-formulated Pc 4 in cells, and significant cytotoxic effect of the formulation upon photoirradiation. Quantitative estimation of the extent of Pc 4 loading in the micelles and the photocytotoxicity of the micelle-incorporated Pc 4 demonstrate the promise of our approach to develop a biocompatible nanomedicine platform for tumor-targeted delivery of Pc 4 for site-selective PDT.

A requirement for bid for induction of apoptosis by photodynamic therapy with a lysosome- but not a mitochondrion-targeted photosensitizer.

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid(-/-) cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes.

Binding to and photo-oxidation of cardiolipin by the phthalocyanine photosensitizer Pc 4.

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT.

A comparative study of the photophysical and phototoxic properties of octakis(decyloxy)phthalocyaninato zinc(II), incorporated in a hydrophilic polymer, in liposomes and in non-ionic micelles.

Factors such as charge, aggregation and lipophilicity influence photosensitiser localisation. The lipophilic octasubstituted sensitiser 2,3,9,10,16,17,23,24-octakis(decyloxy)phthalocyaninato zinc(II) was incorporated into liposomes of dimyristoyl-L-alpha-phosphatidylcholine (DMPC), non-ionic micelles of Tween 80 and the hydrosoluble polymer Solutol HS 15 in order to investigate how these different environments affect the photophysical properties and phototoxicity of the photosensitiser. Fluorescence quantum yields and singlet molecular oxygen generation are enhanced in the presence of Solutol HS 15. Phototoxicities were calculated by employing a concentration of 10(-7) M of the dye against the Hep-2 cell line, which showed a viability of 53 and 30% in DMPC and Solutol HS 15, respectively. After 24 h of photodynamic therapy with 15 min irradiation, apoptotic and necrotic cells were observed.