Borbala Gesser

Cyclin (PCNA, auxiliary protein of DNA polymerase δ) is a central component of the pathway(s) leading to DNA replication and cell division

Cyclin, also known as PCNA or the auxiliary protein of mammalian DNA polymerase δ, is a stable cell cycle regulated (synthesized mainly in S‐phase) nuclear protein of apparent M r 36 000 whose rate of synthesis correlates directly with the proliferative state of normal cultured cells and tissues. Cyclin (PCNA) is absent or present in very low amounts in normal non‐dividing cells and tissues, but it is synthesized in variable amounts by proliferating cells of both normal and transformed origin. All available information indicates that this ubiquitous and tightly regulated DNA replication protein is a central component of the pathway(s) leading to DNA replication and cell division.

IL-8 induces T cell chemotaxis, suppresses IL-4, and up-regulates IL-8 production by CD4+ T cells.

Interleukin‐8 (IL‐8), a neutrophil‐activating cytokine, also activates certain T cell functions such as Chemotaxis. We additionally find (n = 6) that recombinant (rIL‐8; 1–100 ng/ml), when added to 24 h culture of human CD4+ T cells, suppressed the spontaneous production of IL‐4 (50–85%). Steady state production of IL‐4 was typically around 30 pg/ml, determined by use of a solid‐ phase immunoabsorbant assay. De novo synthesis of IL‐4 from CD4+ T cells cultured for 3 days was also evaluated by use of detection of [35S]methionine incorporation, as visualized by autoradiography of 2‐D gels, and showed that IL‐8 suppressed IL‐4 production. This suppression of IL‐4 production was confirmed in the cytosol fraction by use of Western blotting. The effect of IL‐8 (100 ng/ml) was comparable to that of 10 ng/ml recombinant interferon‐γ, both strongly suppressing IL‐4 production. The regulatory effect of IL‐8 on IL‐4 production was also indicated by the fact that addition of a neutralizing monoclonal anti‐IL‐8 antibody (WS.4) enhanced the spontaneous IL‐4 production when added to the cultures of CD4+ T cells, thereby probably inactivating the effect of IL‐8 originating from the cutured T cells. Also, we observed that IL‐4 mRNA expression was down‐regulated when the CD4+ T cells were cultured for 12 h in the presence of 100 ng/ml IL‐8. The suppression of IL‐4 mRNA expression could be prevented by adding anti‐IL‐8 (20 μg/ml) or IL‐10 (100 ng/ml) 1 h before adding rIL‐8. Thus, IL‐8 may be an important regulator of CD4+ T cell‐derived IL‐4, thereby possibly regulating the balance between humoral and cellular T cell‐dependent responses.

Expression of the T‐helper 2‐specific chemokine receptor CCR4 on CCR10‐positive lymphocytes in atopic dermatitis skin but not in psoriasis skin

Background  Atopic dermatitis (AD) and psoriasis are inflammatory skin diseases. AD is generally perceived as a T‐helper (Th) 2‐dominated disease whereas psoriasis is a Th1‐dominated disease. The chemokine cutaneous T‐cell attracting chemokine (CTACK) and its receptor CCR10 attract skin‐homing lymphocytes to inflamed skin, suggesting that CCR10+ cells in AD and psoriasis should be of Th2 and Th1 type, respectively. The chemokine receptor CCR4 is expressed selectively on Th2 lymphocytes and its ligand thymus and activation‐regulated chemokine (TARC) is upregulated in AD lesions, suggesting that the CCR10+ cells in AD lesions should also express CCR4.

Human cellular protein patterns and their link to genome DNA sequence data: Usefulness of two-dimensional gel electrophoresis and microsequencing

Analysis of cellular protein patterns by computer‐aided 2‐dimensional gel electrophoresis together with recent advances in protein sequence analysis have made possible the establishment of comprehensive 2‐dimensional gel protein databases that may link protein and DNA information and that offer a global approach to the study of the cell. Using the integrated approach offered by 2‐dimensional gel protein databases it is now possible to reveal phenotype specific protein (or proteins), to microsequence them, to search for homology with previously identified proteins, to clone the cDNAs, to assign partial protein sequence to genes for which the full DNA sequence and the chromosome location is known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Human 2‐dimensional gel protein databases are becoming increasingly important in view of the concerted effort to map and sequence the entire genome.—Celis, J. E.; Rasmussen, H. H.; Leffers, H.; Madsen, P.; Honoré, B.; Gesser, B.; Dejgaard, K.; Vandekerckhove, J. Human cellular protein patterns and their link to genome DNA sequence data: usefulness of two‐dimensional gel electrophoresis and microsequencing. FASEB J. 5: 2200–2208; 1991.

IL-10 augments the IFN-γ and TNF-α induced TARC production in HaCaT cells: a possible mechanism in the inflammatory reaction of atopic dermatitis

The CC-chemokine TARC is known to be a ligand for the CCR4 receptor which in turn is known to be expressed selectively on the Th(2)-subset of lymphocytes. Atopic dermatitis is generally believed to be a Th(2)-type disease, and TARC has been shown to be expressed in the skin lesions of a murine model of AD. IL-10 is an interleukine generally known for its ability to inhibit cytokine production, however it has been found to be highly expressed in the skin from AD patients. We show in this report that IL-10 is able to augment the TARC inducing effects of TNFalpha and IFNgamma in HaCaT cells, a property that may be important in the determination of the composition of the cells of the inflammation in the skin of AD patients. In addition, we show that the IL10 agonist IT 9302, a nona-peptide from the carboxylic end of IL-10, has the same effect on TARC production from HaCaT cells.

Thymus- and activation-regulated chemokine (TARC/CCL17) induces a Th2-dominated inflammatory reaction on intradermal injection in mice

Abstract:  TARC/CCL17 (thymus‐ and activation‐regulated chemokine) is a CC chemokine, which binds to the CC chemokine receptor‐4 (CCR4) known to be distinctively expressed on Th2 lymphocytes. In atopic dermatitis (AD), the skin is invaded by Th2 lymphocytes in the acute phase. TARC/CCL17 is produced by the keratinocytes in AD lesions, and CCR4 is overexpressed on CLA+ (cutaneous lymphocyte‐associated antigen) lymphocytes in the skin and blood. We, therefore, hypothesized that TARC/CCL17 is pivotal in mediating a Th2‐dominated inflammation in the skin. To examine this, we injected BALB/c mice with murine TARC/CCL17 in concentrations ranging from 0.1 µg/ml to 10 µg/ml and examined the skin after 48 h. This revealed that TARC/CCL17 induces lymphocytic infiltration of the skin by CD4+ lymphocytes in a dose‐dependent manner with a maximum response at 1 µg/ml. Additionally, TARC/CCL17 induced interleukin‐4 mRNA but not interferon‐γ mRNA expression in the skin, suggesting that the lymphocytes invading the skin are Th2 cells. Additionally, TARC/CCL17 induced its own production in the keratinocytes along with cutaneous T‐cell‐attracting chemokine (CTACK/CCL27) mRNA. We, therefore, conclude that TARC/CCL17 induces a Th2‐dominated inflammatory reaction when injected into the skin.

Protein-Electroblotting and Microsequencing in Establishing Integrated Human Protein Databases

Proteins, which are characteristic for a specific state of differentiation, the transformed phenotype or pathological conditions of human cells and tissues were identified by computer analyzed two-dimensional gel electrophoresis. Sequenceable amounts of protein were collected from multiple gels with a gel-concentration device, enabling the elution and concentration of more than twenty protein spots, suspended in 1 ml of sample buffer. The eluted protein was concentrated in a new gel in a very small spot and then electroblotted onto polybase-coated glass-fiber or polyvinylidene-difluoride membranes and in situ digested. The released peptides were separated by micro-bore or narrow-bore reversed phase HPLC and immediately collected on polyethylenimine-coated glass-fiber discs for sequencing. These variations of previously developed methods allowed us to work at higher sensitivity. The procedure is currently being used to try out a systematic analysis of human proteins recovered from two-dimensional gels.

Cytokine expression of skin T-lymphocytes from patients with atopic dermatitis

We analysed the cytokine profile of skin T cells by establishing 11 T-cell lines from adult patients with moderate-to-severe atopic eczema using T-cell growth factors interleukin-2 and interleukin-4. We compared T-cell lines from lesional skin of atopic dermatitis patients with those from non-atopic skin of patients with other skin diseases, observing that T-cell lines of patients with atopic dermatitis unstimulated cultures expressed a Th1 profile. After stimulation with anti-CD3 and anti-CD28 monoclonal antibodies, the cytokine expression showed rapid initial upregulation of Th2 followed by a Th1 profile. Furthermore, strong upregulation of interleukin-10 was observed after 24 h stimulation. Our findings suggest that skin T-lymphocytes from atopic dermatitis patients seem to consist of a heterogenous population of Th1 and Th2 or Th0 cells and the results for secreted cytokines indicate that T-cell lines from each inflammatory skin disease showed the corresponding disease-specific original cytokine profile.

Routine Amino Acid Sequencing on 2D-Gel Separated Proteins: A Protein Elution and Concentration Gel System

2D-gel electrophoresis is routinely used to study the integral protein composition of a given cell type, often revealing more than 2,000 individual spots in a single gel. Due to its high resolution power, the technique has been employed to study alterations in cellular protein expression in response to various stimuli or as a result of differentiation and development (Celis and Bravo, 1984). Identification of the separated proteins can be achieved by comigration electrophoresis or by immunological techniques. Proteins can also be recognized by their amino acid composition, their exact molecular weight as determined by mass spectrometry or by partial amino acid sequencing. The latter approach further allows cDNA cloning from the resultant sequences.

Comprehensive, human cellular protein databases and their implication for the study of genome organization and function

Comprehensive, computerized databases of cellular protein information derived from the analysis of two‐dimensional gels, together with recently developed techniques to microsequence proteins offer a new dimension to the study of genome organization and function. In particular, human protein databases provide an ideal framework in which to focus the human genome sequencing effort.