Effect of partially modified retro‐inverso analogues derived from C‐reactive protein on the induction of nitric oxide synthesis in peritoneal macrophages

The ability of three modified tetrapeptides, representing fragments of the C‐reactive protein (CRP) sequence and stabilized in the first peptide bond by retro‐inverso modification, to affect the secretion of nitric oxide (NO) was studied in macrophages of BALB/c mice. These tetrapeptides, resembling the aminoacid sequence of tuftsin (CRP I, H‐gThr‐(R,S)mLys‐Pro‐Leu‐OH, ITF 1192; CRP II, H‐gGly‐(R, S)mLys‐Pro‐Arg‐OH, ITF 1127; CRP III, H‐gThr‐(R,S)mLys‐Pro‐Gln‐OH, ITF 1193), were able to induce NO synthesis by peritoneal macrophages in a dose‐dependent manner; the most stimulating dose was 1000 ng ml−1 for CRP II and 100 ng ml−1 for CRP I and CRP III. NO synthesis was not strictly dependent on lipopolysaccharide (LPS) activation. The enhanced effect of retro‐inverso CRP‐related analogues on the expression of iNOS (inducible NO synthase) was confirmed by higher levels of iNOS activity in the cytosol and by the increase in iNOS protein, as evaluated by Western blot analysis, in macrophages stimulated by CPR compared with untreated ones. The production of NO by retro‐inverso CRP‐peptide analogues was significantly inhibited by dexamethasone (20 μm), NG‐monomethyl‐l‐arginine (l‐NMMA) (500 μm) and pyrrolidine dithiocarbamate (PDTC) (100 μm). Retro‐inverso CRP‐peptide analogues stimulated macrophages to produce high levels of interleukin‐1 (IL‐1) and tumour necrosis factor‐α (TNF‐α) in the presence of LPS. Retro‐inverso CRP‐peptide analogues stimulated NO synthesis by the enhancement of endogenously produced IL‐1 and TNF‐α, as the treatment of peritoneal macrophages with LPS in the presence of neutralizing anti‐IL‐1 and anti‐TNF monoclonal antibodies (mAbs) reduced retro‐inverso analogue‐induced NO secretion. Data indicate a predominant role for IL‐1α in the induction of NO secretion by retro‐inverso analogues. These results suggest that retro‐inverso CRP derived analogues act as costimulators of NO and cytokine synthesis in macrophages. The mechanisms by which they cause iNOS induction appear to be strongly dependent on the activation of nuclear factor‐κB (NF‐κB).

[1]  J. Lancaster,et al.  Synthesis of nitric oxide from a terminal guanidino nitrogen atom of L-arginine: A molecular mechanism regulating cellular proliferation that targets intracellular iron , 1990 .

[2]  K. Nishioka,et al.  Effect of Tuftsin on the Migration, Chemotaxis, and Differentiation of Macrophages and Granulocytes a , 1983, Annals of the New York Academy of Sciences.

[3]  M. Krönke,et al.  Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling , 1994, Cell.

[4]  G. Gromo,et al.  The macrophage-activating tetrapeptide tuftsin induces nitric oxide synthesis and stimulates murine macrophages to kill Leishmania parasites in vitro , 1994, Infection and immunity.

[5]  J. Drapier,et al.  Interferon‐γ and tumor necrosis factor induce the L‐arginine‐dependent cytotoxic effector mechanism in murine macrophages* , 1988 .

[6]  P. Gottlieb,et al.  Tuftsin, Thr-Lys-Pro-Arg , 1981 .

[7]  C. Nathan,et al.  Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. , 1988, Journal of immunology.

[8]  M. Salter,et al.  Widespread tissue distribution, species distribution and changes in activity of Ca2+‐dependent and Ca2+‐independent nitric oxide synthases , 1991, FEBS letters.

[9]  A. Baldwin,et al.  THE NF-κB AND IκB PROTEINS: New Discoveries and Insights , 1996 .

[10]  P. Baeuerle,et al.  Function and activation of NF-kappa B in the immune system. , 1994, Annual review of immunology.

[11]  I. Kushner,et al.  SERUM C‐REACTIVE PROTEIN LEVELS IN DISEASE * , 1982, Annals of the New York Academy of Sciences.

[12]  G. Rovati,et al.  Lower efficacy: interaction with an inhibitory receptor or partial agonism? , 1994, Trends in pharmacological sciences.

[13]  M. Witko,et al.  Quantum-chemistry and catalysis in oxidation of hydrocarbons , 1981 .

[14]  G. Nabel,et al.  Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T. Lint,et al.  Interaction of C-reactive protein with lymphocytes and monocytes: complement-dependent adherence and phagocytosis. , 1976, Journal of immunology.

[16]  P. Baeuerle Dithiocarbamates as Potent Inhibitors of Nuclear Factor KB Activation in Intact Cells By galf Schreck, Beate Meier,* Daniela N. M~innel,~ Wulf Dr6ge,~ , 1992 .

[17]  M. Fridkin,et al.  The effect of Tuftsin on the nitrous blue tetrazolium reduction of normal human polymorphonuclear leukocytes. , 1975, The Journal of clinical investigation.

[18]  K. Nishioka,et al.  Tuftsin: a hormone-like tetrapeptide with antimicrobial and antitumor activities. , 1981, Life sciences.

[19]  B. Fiedel Influence of tuftsin-like synthetic peptides derived from C-reactive protein (CRP) on platelet behaviour. , 1988, Immunology.

[20]  B. Brenner,et al.  Interleukin 1 induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. , 1991, The Journal of clinical investigation.

[21]  M. Fridkin,et al.  Tuftsin: its chemistry, biology, and clinical potential. , 1989, Critical reviews in biochemistry and molecular biology.

[22]  V. Pliska Models to explain dose-response relationships that exhibit a downturn phase. , 1994, Trends in pharmacological sciences.

[23]  A. Levitzki,et al.  Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors. , 1994, Science.

[24]  R. Heuertz,et al.  Peptides derived from C-reactive protein inhibit neutrophil alveolitis. , 1996, Journal of immunology.

[25]  M. Pepys,et al.  Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein. , 1983, Advances in immunology.

[26]  S. Futaki,et al.  Proteolysis of human C-reactive protein produces peptides with potent immunomodulating activity. , 1987, The Journal of biological chemistry.

[27]  P. Libby,et al.  Induction and Stabilization of IκBα by Nitric Oxide Mediates Inhibition of NF-κB (*) , 1995, The Journal of Biological Chemistry.

[28]  W. Greene,et al.  Tumor necrosis factor alpha induces proteins that bind specifically to kappa B-like enhancer elements and regulate interleukin 2 receptor alpha-chain gene expression in primary human T lymphocytes. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[29]  D. Männel,et al.  Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells , 1992, The Journal of experimental medicine.

[30]  K. Nishioka,et al.  Prophylaxis of Candida albicans infection with tuftsin. , 1986, Journal of Antimicrobial Chemotherapy.

[31]  P. Majerus,et al.  Inositol Hexakisphosphate Binds to Clathrin Assembly Protein 3 (AP-3/AP180) and Inhibits Clathrin Cage Assembly in Vitro(*) , 1995, The Journal of Biological Chemistry.

[32]  K. Nishioka,et al.  ‘Tuftsin’: a Natural Phagocytosis Stimulating Peptide , 1970, Nature.

[33]  I. Kushner,et al.  C-reactive protein and the acute-phase response. , 1990, Hospital practice.

[34]  T. Mosmann Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[35]  Murray Goodman,et al.  On the Concept of Linear Modified Retro-Peptide Structures , 1979 .

[36]  C. Nathan,et al.  Identification of arginine as a precursor of endothelium-derived relaxing factor. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Longobardi,et al.  Immunostimulation by a partially modified retro-inverso-tuftsin analogue containing Thr1 psi[NHCO](R,S)Lys2 modification. , 1991, Journal of medicinal chemistry.

[38]  B. Huber,et al.  Interleukin‐1 induces c‐fos and c‐jun gene expression in T helper type II cells through different signal transmission pathways , 1992, European journal of immunology.

[39]  R. Busse,et al.  Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells. , 1995, Circulation research.

[40]  F. Bonelli,et al.  Solid phase synthesis of retro-inverso peptide analogues. Synthesis and biological activity of the partially modified retro-inverso analogue of the bradykinin potentiating peptide BPP9a [gLys6, (RS)-mPhe7, Ala8] BPP9a. , 2009, International journal of peptide & protein research.

[41]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[42]  J. Robotham,et al.  The acute-phase response. , 1995, New horizons.

[43]  Z. Fuks,et al.  Toxicology and Antitumor Activity of Tuftsin a , 1983, Annals of the New York Academy of Sciences.

[44]  J. Volanakis,et al.  Probing the C1q-binding site on human C-reactive protein by site-directed mutagenesis. , 1994, Journal of immunology.

[45]  A. Whitehead,et al.  The major acute phase reactants: C-reactive protein, serum amyloid P component and serum amyloid A protein. , 1994, Immunology today.

[46]  P. Kaumaya,et al.  Cell attachment peptide of C‐reactive protein: critical amino acids and minimum length , 1994, Journal of cellular biochemistry.

[47]  M. Ginsberg,et al.  Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. , 1991, Trends in biochemical sciences.

[48]  S. Moncada,et al.  Vascular endothelial cells synthesize nitric oxide from L-arginine , 1988, Nature.