UdA CYTOKINES AND CHEMOKINE IN INFLAMMATION: Dr. P. Conti - - PowerPoint PPT Presentation

Professor Pio Conti Director of the Division of Immunology University of Chieti ITALY

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CYTOKINES AND CHEMOKINE IN INFLAMMATION: Dr. P. Conti

• Cytokine

are proteis secreted by the cells

• f

innate and adaptive immunity that mediate many

• f the functions
• f these

cells.

• Cytokines

are produced in response to microbes and other antigens.

• Different

cytokine stimulate diverse responses

• f

cells involved in immunity and inflammation.

• Nomenclature:monokines, lymphokines,

cytokines, interleukines, chemokines.

• Cytokines

are pleiotropic and redundantand and they also have an autocrine action.

T and B LYMPHOCYTES

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Professor P. Conti Division of Immunology University of Chieti - ITALY

Macrofago presentante l’antigene (APC) CD4 o CD8 CD3 MHC Antigene Linfocita T Producing Cytokines & Chemokines TCR

T-cell activation

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B and T-cell activation by the antigen

Professor P. Conti Division of Immunology University of Chieti - ITALY Ag macrofago Fagocitosi dell’Ag Macrofago attivato Antigene processato Recettore per l’Ag Linfocita T Linfocita B IL-1 Presentazione dell’Ag al linfocita B IL-1 macrofago Linfocita B Clone cellulare Linfocita T attivato Clone cellulare Linfocita B attivato TCR/CD3 Ag MHC

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Cytokines and inflammatory cells starting from stem cells

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Hypersensitivity Type IV

Activated lymphocyte produce cytokines:actvation of macrophage and release of inflammatory monokines.

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Comparison of the biologic properties of monokines: IL-1, TNF and IL-6

Professor P. Conti Division of Immunology University of Chieti - ITALY

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BIOLOGICAL EFFECTS OF IL-1 Aspects of the acute phase response mediated by IL-1

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-1 downregulation by IL-4, IL-6, PGE and IL-1ra (network)

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IL-6 and B lymphocyte activation IL-6 binds B cell IL-6Ra

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-6 production and its involvement in disease

Professor P. Conti Division of Immunology University of Chieti - ITALY

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Maturation of mast cells and IL-6

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-32

Manuscript n preparation 2007-2008 IL-32α A PRO-INFLAMMATORY CYTOKINE IN RAT PAW INJECTION SITE ACTIVATES COX-2 GENE EXPRESSION AND STIMULATES PGE2 GENERATION : LACK OF EFFECT ON PGD2 , HDC mRNA EXPRESSION, HISTAMINE AND TRYPTASE RELEASE IN CORD BLOOD MAST CELLS

1Castellani M.L., 2

Kempura D.J, 2Boucher W., 2Tagen M., 3Frydas S., 4Perrella A., 2Theoharides T.C., 5Neri G.,

6Cerulli G. and 1Conti P.

1Immunology Division, Medical School, University of Chieti-Pescara, Italy; 2Department of Pharmacology and Experimental Therapeutics,

Biochemistry and Internal Medicine Tufts University School of Medicine, Tufts-New England Medical Center, Boston, MA, USA ; 3Laboratory

• f Parasitology, Veterinary Faculty, Aristotele University, Thessaloniki, Greece. 4Infectious Diseases, University of Napoli, Italy.

5ENT Division, University of Chieti-Pescara, and 6Orthopaedic Division, University of Perugia, Italy

Interleukin-32 (IL-32) is one of the latest described inflammatory cytokine, closely related to IL-1, although, the structure and the receptors are clearly distinct. IL-32 is produced by T cells, NK cells, epithelial cells and monocytes/macrophages and its transcripts are highly expressed in immune tissues. IL-32 is a pro-inflammatory cytokine capable to induce other pro-inflammatory cytokines, such as IL-1, IL-6, TNF-alpha and IL-8 (CXCL8).

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-32 inflammatory effect on the rat paw

PBS IL-32 Anti-IL-32 Anti-IL-32 + IL-32

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-32 failed to stimulate tryptase release in human mast cells

Tryptase production from human cord blood-derived mast cells Tryptase (mg/ml) Three experiments in triplicate Groups 1 2 3 Control 2.3 ± 0.5 2.0 ±0.6 2.1 ± 0.7 Anti-IgE 10μg/ml 510 ± 80 480 ± 110 390 ± 90 IL-32α 20μg/ml 1.8 ± 0.3 2.4 ± 0.9 1.9 ± 0.6 IL-32α 50μg/ml 1.7 ± 0.5 2.1 ± 0.6 2.2 ± 0.8

Professor P. Conti Division of Immunology University of Chieti - ITALY

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The biology of IL-33: the latest interleukin

• IL-33 is the natural ligand of the IL-1 receptor family member ST2L
• IL-33 is involved in allergic inflammatory responses through the production of

cytokines released by Th2 cells.

• IL-33 stimulates mast cell to produce pro-inflammatory cytokines and mediates

allergic disorders.

• IL-33 accelerates the maturation of CD34+ mast cell precursor of human cord

blood-derived mast cells.

• IL-33 induces the production of Th2 cytokines and is produced in the sera of

asthmatic patients.

• IL-33 is a selective Th2 chemoattractant.
• IL-33 mediates IL-8 production by human cord blood mast cells.

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Expression and secretion of rantes (CCL5) in granulomatous calcified tissue before and after lipopolysaccharide treatment In vivo We induced an experimental chronic inflammatory state in rats by subcutaneous injection (0.2 ml) of a saturated water solution (1:40) potassium permanganate (KMnO4 ). This treatment causes the formation of calcified granulomatous tissue at the site of injection, reaching the apex in size and weight after one week.

Professor P. Conti Division of Immunology University of Chieti - ITALY

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RANTES IN GRANULOMA

RANTES concentration (pg/ml/100 mg), determined by ELISA, in the conditioned medium from minced granuloma tissue induced in rats with KMnO4 and i.p.-treated in vivo with LPS or Dex or PBS. LPS (100 ng/ml) or nothing was added in vitro in all specimens from previously i.p.-treated animals.

RANTES pg/ml/100 mg tissue Treatment in vivo

• No. of

animals

• LPS

in vitro +LPS in vitro P<0. 05 Δ % PBS (control) 12 455 ± 82 1430 ± 275 (0.05 ) + 214.28 % LPS 12 1680 ± 395 3560 ± 195 (0.01 ) +111.9 % Dex 12 337 ± 20 570 ± 150 (N.S. ) + 69.1 % %

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IL-10 network

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-10 immunostimulatory effects

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-10 and mast cells

Professor P. Conti Division of Immunology University of Chieti - ITALY

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Chemokines (I)

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Chemokines (II)

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Chemokines (III)

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Dermal response to exogenous human RANTES

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Mast cell migration induced by RANTES

Professor P. Conti Division of Immunology University of Chieti - ITALY

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RANTES and HDC

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IL-19, IL-20, IL-22, IL-24, and IL-26 network and biological effects

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-19 network

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-21 network

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-22

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-25

Professor P. Conti Division of Immunology University of Chieti - ITALY

Professor P. Conti Division of Immunology University of Chieti - ITALY

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HIV infection of CD4+ lymphocytes

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Tumor and cytokines

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Tumor and cytokines

Professor P. Conti Division of Immunology University of Chieti - ITALY

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Professor P. Conti Division of Immunology University of Chieti - ITALY

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Differential production of IL-1 and IL-1 receptor antagonist (IL-1Ra)

Professor P. Conti Division of Immunology University of Chieti - ITALY

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IL-15 binds the IL-12 receptor

IL-15 IS STRUCTURAL HOMOLOGUS TO IL-2

• IL-15 IS A CYTOKINE PRODUCED BY

MONONUCLEAR CELLS IN RESPONSE TO VIRAL INFECTION, LPS AND OTHER SIGNALS.

• IL-15 ACTS A T-CELL GROWTH FACTOR ON

LONG LIVED MEMORY CD8+ T CELLS.

• IL-15 KNOCKOUT MICE HAVE GREATLY

REDUCED NUMBERS OF NK AND CD8+ CELLS.

• IL-15 network

IL-15 (TCGF)

IL-1β TNFα chemokines

• CD8+ T-cell
• NK cell
• DC
• neutrophil
• macrophage

T & B cell

• keratinocyte
• melanoma cell

Inflammation

production

Mast cell IL-4 Th2 response

development growth survival homeostasis (anti-tumor)

FUNCTION stimulation

apoptosis release

DC:

• Antigen

presentation

• IL-2 production

NK / T-cell:

• Cytolotic

activity

• KIR upreguklation

IL-15 AN IMMUNOREGULATORY AND ANTI-CANCER CYTOKINE: RECENT ADVANCES L.N. SHANMUNGAM 1, C. PETRARCA2, S. FRYDAS 3, J. DONELAN4, M.L. CASTELLANI 5, W. BOUCHER4, B. MADHAPPAN4, S. TETE’6, K. FALASCA7, J. VECCHIET7 and P. CONTI2 1Department of Radiation Oncology, Brigham and Women's Hospital,HARVARD MEDICAL SCHOOL, Boston, MA 02115, U.S.A.; 2Division

• f Immunology, 6Orthopaedics and 7Infection Disease, University of Chieti,

Italy; 3Department of Parasitology, Veterinary School, Aristotelian University, Thessaloniki, Greece; 4Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, U.S.A

IL-2

IL-2Rα IL-2Rβ IL-2Rγ

IL-15

IL-15Rα IL-2Rγ IL-2Rβ

NUCLEUS

• Time-course of PGD2 production by RBL cells

upon activation by RANTES.

1 2 3 4 0,5 1 2 4 8 12 time (hours) PGD2 (ng/ml)

Treatment PDG2 (ng/ml) Control (PBS) 0.2 ± 0.2 A23187 4.7 ± 1.0 RANTES (0.2 ng/ml) 0.3 ± 0.2 RANTES (2 ng/ml) 2.0 ± 0.9 RANTES (20 ng/ml) 3.9 ± 0.8 RANTES (100 ng/ml) 4.2 ± 0.7 RANTES (200 ng/ml) 4.1 ± 0.9

Generation of PDG2 by RBLs after RANTES treatment (scalar concentration doses).

RBL cells produce PDG2 upon activation by RANTES, which can be abrogated by indomethacine.

1 2 3 4 5 6 control Indom ethacine RANTE S A23187 Indomethacine + A23 Indom ethacine + RANTE S PGD2 (ng/ml)

• Rat basophilic leukaemia (RBL) cells release PDG2 in the culture

medium after treatment with RANTES.

1 2 3 4 5 6 control (PBS) A23187 RANTES 0.2 ng/ml RANTES 2.0 ng/ml RANTES 20 ng/ml RANTES 100 ng/ml RANTES 200 ng/ml PDG2 (ng/ml)

a) Mast cellula a riposo; b) mast cellula dopo venti secondi dall'inizio della degranulazione (microscopio elettronico a trasmissione). Le frecce indicano il punto della de granulazione (fig. B).

Le IgE, al contrario di molti altri anticorpi, possono essere inattivate alla temperatura di 56°C per 1 ora. Esse non attraversano la placenta e non si legano al complemento, ma hanno nella loro catena pesante una struttura che le fa reagire con i recettori presenti sulla superficie delle mast cellule e dei granulociti basofili. Per provocare una reazione allergica sulla pelle, sono richiesti solo 0.1-0.001 ug/ml di anticorpi IgE specifici. Quindi le IgE sono Le mast cellule hanno più di 100.000 recettori per cellula.

• Ipersensibilità di tipo II °: citotossica
• Le IgG

possono legarsi agli Ag della membrana cellulare della cellula bersaglio (per esempio globulo rosso incompatibile) attivando una reazione citotossica mediata dalle cellule "killer" o dal complemento, con conseguente lisi della cellula bersaglio.

• IN THIS REACTION, LYMPHOCYTES PRODUCE SEVERAL IMPORTANT

CYTOKINES THAT HELP TO DEMAGE THE RED BLOOD CELLS

• Ipersensibilità di tipo III: da immunocomplessi
• Gli immunocomplessi

si depositano nei tessuti e si attiva il complemento. La deposizione degli immunocomplessi richiama i granulociti polimorfonucleati (PMN) i

• quali provocano dei danni "in loco".
• complement
• Tissue
• PMN producing cytokines and chemokines CXC

IL-5

• IL-5 is

an activator

• f eosinophils

and serves as a link between T cell activation and and eosinophilic inflammation.

• It

is produced by the Th2 subset of CD4+ T cellsand by activated mast cells.

• The two

main Th2 cytokines, IL-4 and IL-5 function in concert: IL-4 stimulates the production of IgE, which

• psonizes

helminths, and binds eosinophils, and IL-5 activates the eosinophils to destroy the parasites.

• IL-5 stimulates

the proliferation

• f B cells

(IgA)

TGF-β, IL-7 and IL-3

• The principal

action of TGF-β in immunity is to inhibit the proliferation and activation of lymphocytes and other leukocytes.

• There are three TGF-β: TGF-β1, TGF-β2, TGF-β.
• The immune system synthesize mainly TGF-β1.
• Some Reg. Τ cells produce TGF-β

and the same produce also IL-10. ============================================

• IL-7 is secreted by stromal cells in many tissues. IL-7 stimulates

survival and expansion of immature precursor committed to the B and T lymphocyte lineages.

• IL-7 is essential for the survival of immature, naive T cells and

memory cells, especially CD4+ memorycells.

• ==================================================================I
• ΙL-3 is a multilineage colony stimulating factor (multi-CSF) and is

produced by CD4+ T cells.

• ΙL-3 promotes the growth and differentiation of mast cells from bone

marrow-derived progenitors.

IL-13, IL-16, and IL-17

• IL-13 is

a cytokine similar to IL-4 that is produced by Th2 CD4+ T cells and by some epithelialcells.IL-13 receptor is found mainly

• n macrophages

(activation).

• IL-13 appears

to have less effect

• f IL-4 in T and B

lymphocytes.

• ===============================================
• IL-16 is

a T cell-derived cytokine that acts a specific chemoattractant

• f eosinophils.
• ===============================================
• IL-17 consist
• f a family of cytokinesthat

are produced by activated and memory T cells and induce the production of

• thr

proinflammatory cytokines, such as TNF, IL-1, and chemokines.

microRNA

• Contemporary biology has been revolutionized by a recently discovered

class of small regulatory RNA molecules, microRNAs (miRNAs). miRNAs has been missed by researchers for decades due to their tiny size, usually mapping to non-protein-coding regions of genomes.

• microRNAs (1-5% of all genes) are a class of small RNAs, noncoding

RNAs, that are increasingly being recognized as important regulators of gene expression and are involved in biological control at multiple levels and regulate gene expression at the posttranscriptional level.

• Although hundreds of microRNAs are present in the mammalian

genome, genetic studies addressing their physiological roles are at an early stage.

• microRNAs (miRNAs) are a class of 19- to 23-nt, small, noncoding

RNAs, which bind the 3' UTR of target mRNAs to mediate translational repression in animals. miRNAs have been shown to regulate developmental processes, such as self-renewal of stem cells, neuronal differentiation, myogenesis, and cancer. A functional role of miRNAs in the regulation of neurotransmitter synthesis has yet to be ascribed.

• microRNAs and siRNAs are small noncoding RNAs, that regulate gene

expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi.

• microRNAs are natural, single-stranded, small RNA molecules that regulate gene

expression by binding to target mRNAs and suppress its translation or initiate its degradation.

• microRNAs (miRNAs) are a class of small regulatory RNAs that are thought to regulate

the expression of as many as one-third of all human messenger RNAs (mRNAs). miRNAs are thought to be involved in diverse biological processes, including tumorigenesis. Analysis of miRNA levels may have diagnostic implications. Evidence shows that numerous viruses interact with the miRNA and that a number of viruses encode their

• wn miRNAs. It seems likely that miRNAs will be implicated in many human diseases.

Manipulation of miRNA levels by gene therapy provides an attractive new approach for therapeutic development.

• Expression profiling in vivo demonstrates rapid changes in lung microRNA levels

following lipopolysaccharide-induced inflammation but not in the anti-inflammatory action of glucocorticoids. At present, nothing is known of the role of miRNAs in the immune response in vivo despite the fact that inflammation is thought to underlie multiple acute and chronic diseases. In these circumstances, patients are commonly treated with corticosteroids such as dexamethasone.

• Moschos SA, et al. shown that the LPS-induced innate immune response is associated

with widespread, rapid and transient increases in miRNA expression in the mouse lung and we speculate that these changes might be involved in the regulation of the inflammatory response. In contrast, the lack of effect of dexamethasone in either control

• r challenged animals implies that the actions of glucocorticoids per se are not mediated

through changes in miRNAs expression and that LPS-induced increases in miRNA expression are not mediated via classical inflammatory transcription factors.

• microRNAs (miRNAs) have emerged as a class of gene expression

regulators that has also been linked to cancer and inflammation.

• Wu X., et al., reported that downregulation of CCR1 inhibits human

hepatocellular carcinoma cell invasion. CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of hepatocellular carcinoma cells.

• microRNA-155 plays a key role in the homeostasis and function of the

immune system.

• Individual microRNAs can exert critical control over mammalian

differentiation processes in vivo.

• Regulatory T (T reg) cells have a miRNA profile distinct from

conventional CD4 T cells. Depleting miRNAs reduces T reg cell numbers and results in immune pathology. miRNAs facilitate, in a cell- autonomous fashion, the development of T reg cells in the thymus and the efficient induction of Foxp3 by transforming growth factor beta.

RNA Interference (RNAi)

• The Nobel Prize in Physiology or Medicine was awarded in 2006 to Andrew Fire

(Stanford University School of Medicine) and Craig Mello (University of Massachusetts Medical School) for their discovery of a new form of gene

• silencing. Nearly 9 years ago, Fire and Mello and their colleagues reported that

exposing cells of nematode to double-stranded RNA resulted in specific and efficient gene silencing. They also observed that double-stranded RNA is far more potent than sense or antisense RNA in silencing the gene that shares its sequence, and they dubbed the silencing process "RNA interference" (RNAi). (siRNA) is called "small interfering RNA".

• In lower organisms, RNAi is thought to function as a primitive immune system,

protecting against viruses (which often generate double-stranded RNA as replication intermediates) and transposable elements (also known as "jumping genes").

• In most mammalian cells, long double-stranded RNA provokes an interferon

response as part of an antiviral defense.

• RNAi has many applications in biomedical research, including drug

development.

• RNAi can be used to identify genes involved in drug resistance
• Many diseases are caused by the inappropriate activity of specific genes, and

the selective silencing of such genes through RNAi represents a potential therapeutic strategy for such diseases.

microRNAs and PSORIASIS

• Psoriasis-affected skin has a specific microRNA

when compared with healthy human skin or with

• ther skin disease.
• miR-203 shows a highly skin-specific expression

profile and is up-regulated in psoriac plaques.

• microRNA

is involved in pathogenesis of psoriasis and contributes to the dys- function of cross talk between resident and infiltrating cells.