A monoclonal antibody to the IL-1 beta peptide 163-171 blocks adjuvanticity but not pyrogenicity of IL-1 beta in vivo

The synthetic fragment VQGEESNDK, corresponding to the amino acid sequence in position 163-171 of human IL-1 beta, possesses the immunostimulatory but not the pyrogenic activity of the mature IL-1 beta polypeptide in vivo. To assess the relevance of this domain of IL-1 beta for its biologic activities, a mAb was raised against the synthetic peptide 163-171. The mAb Vhp20 could effectively recognize human rIL-1 beta in RIA and immunoblotting. In vivo, the mAb Vhp20 was able to selectively inhibit the immunostimulatory activity of IL-1 beta, but it could not affect the fever-inducing capacity of IL-1 beta. It is proposed that functional domains could be identified in the human IL-1 beta protein and that the fragment in position 163-171 is of major importance for the adjuvant capacity of the entire molecule, but irrelevant to its pyrogenic activity.     

In vivo immunostimulating activity of the 163-171 peptide of human IL-1 beta

The stimulating effect of a synthetic nonapeptide (fragment 163-171) of human interleukin 1 beta (IL-1 beta) on antibody responses to both T helper-dependent and T helper-independent antigens was investigated. It was shown that the nonapeptide enhanced the antibody response, as evaluated in the hemolytic plaque assay, of spleen cells from mice immunized with sheep red blood cells (SRBC). The activity of the 163-171 peptide on the primary response to SRBC was dose-dependent, being maximal when the peptide was inoculated at 100 mg/kg together with the antigen. Moreover, the 163-171 peptide was also effective in enhancing the secondary response to SRBC. The effect of the 163-171 peptide was to augment the frequency of cells specific for the antigen, inasmuch as no increase was ever observed in spleen cell numbers after treatment. In all these studies, human recombinant IL-1 beta gave effects qualitatively comparable to those of the 163-171 peptide, with a maximal activity at 20 ng/kg. Both the 163-171 peptide and human recombinant IL-1 beta were also able to enhance the in vivo immune response to a T helper-independent antigen such as SIII, a poorly immunogenic polysaccharidic antigen from Streptococcus pneumoniae type III. It can therefore be proposed that this synthetic nonapeptide of human IL-1 beta may represent a good candidate for use as adjuvant in vaccines.     

Defining the structural requirements of a biologically active domain of human IL-1 beta

The immunostimulatory activity in vivo of the pleiotropic cytokine IL-1 beta can be retained by its nonapeptide VQGEESNDK, in position 163-171. A series of shorter and longer peptides around this position has been assayed for IL-1-like biological activity, in order to identify the structural requirements for full expression of adjuvant capacity. Elongated peptides, comprising the loop region 165-169 and up to six amino acids in the preceding beta strand or up to seven amino acids in the following beta strand, showed activity comparable or lower than that of the nonapeptide 163-171. This would indicate that the beta strand sequences are not required for optimizing the active conformation of the immunostimulatory IL-1 beta moiety. Accordingly, stabilization of the 163-171 peptide conformation by cyclization did not increase its biological activity. In contrast, the pentapeptide GEESN, corresponding the exposed loop 165-169 between two beta strands, had biological activity higher than that of the 163-171 nonapeptide and fully comparable to that of the entire IL-1 beta protein. Thus, the highly exposed fragment 165-169 within the IL-1 beta molecule may be the structure selectively responsible for the IL-1 beta immunostimulatory capacity in vivo.     

Comparison of human interleukin-1 beta and its 163-171 peptide in bone resorption and the immune response

Human interleukin-1 beta (IL-1 beta) caused a dose- and time-dependent enhancement of the release of 45Ca from prelabeled mouse calvaria in organ culture. In addition, IL-1 beta dose-dependently stimulated the formation of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha in the calvarial bones. However, IL-1 beta-induced 45Ca release was only partially inhibited by blocking the PGE2 response with indomethacin, suggesting that enhanced PGE2 formation in response to IL-1 beta is not necessary to obtain a bone resorptive effect, but that prostaglandins potentiate the action of IL-1 beta. The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, administered simultaneously with antigen (SRBC) to C3H/HeN male mice, induced a dose-dependent enhancement of specific antibody-producing cells in the spleen (PFC). The degree of PFC stimulation was comparable to that caused by native human IL-1 beta. In mouse bone cultures, neither 45Ca release nor prostanoid formation was stimulated by fragment 163-171. These data indicate that (1) IL-1 beta-induced stimulation of bone resorption is dissociable from IL-1 beta-induced increase of prostanoid biosynthesis and (2) the epitope of the IL-1 beta molecule involved in the immunostimulatory effects may be different from that involved in the stimulatory effects on bone resorption.     

In vivo restoration of T cell functions by human IL-1 beta or its 163-171 nonapeptide in immunodepressed mice

The immunorestorative capacities of human (hu) IL-1 beta or its synthetic fragment 163-171 (VQGEESNDK) were assessed in vivo in mice immunodepressed by aging, sublethal irradiation, or both. Subcutaneous administration of hu rIL-1 beta into immunodepressed animals immediately after carrier (horse red blood cells, HRBC) priming could restore to normal levels Th cell activity. This was measured as the ability of spleen cells from HRBC-primed mice to induce a hapten-specific antibody response in spleen cells from nonimmune mice in vitro stimulated with the hapten-carrier conjugate TNP-HRBC. In parallel, the ability of spleen cells from hu rIL-1 beta-treated immunodepressed animals to produce T cell growth factor activity upon in vitro mitogen stimulation was also increased significantly as compared to that of untreated mice and approached that of immunocompetent controls. The immunorestorative activity of hu rIL-1 beta on Th cell activity and T cell growth factor production could be mimicked by the synthetic nonapeptide 163-171 which, at the doses used, produced in most instances even greater effects than the whole protein. Although the optimal immunorestorative doses of the 163-171 peptide were several orders of magnitude higher than those of hu rIL-1 beta, the complete lack of IL-1-like inflammatory and toxic effects suggests that the synthetic hu IL-1 beta fragment may be successfully used as immunomodulating agent in the therapy of T cell immunodeficiencies.     

Lymphokine-activated tumor inhibition in mice. Ability of a nonapeptide of the human IL-1 beta to recruit anti-tumor reactivity in recipient mice

The ability of the VQGEESNDK synthetic peptide corresponding to fragment 163-171 of human IL-1 beta to trigger lymphokine-activated tumor inhibition (LATI) of a poorly immunogenic fibrosarcoma (CE-2) of BALB/c mice was compared to that of the whole IL-1 beta. Neither molecule inhibits in vitro proliferation of CE-2 cells. Administration at the tumor challenge site for 10 days of daily injections of 50 micrograms of peptide 163-171 induce a consistent, although limited, inhibition of tumor growth, whereas similar injections of 1 pg of IL-1 beta induced a more marked LATI. However, strong LATI was elicited when these injections were performed in mice challenged with tumor cells admixed at 1/10 cell ratio with nonreactive lymphocytes from CE-2-bearing mice. The L3T4+ lymphocyte subset is mainly responsible for this enhancement. This reaction is abolished when recipient mice are sub-lethally irradiated, treated with cyclosporin A, or when the reactivity of L3T4+ and asialo GM1+ cells is suppressed. A similarly efficient LATI is found on combining the daily peptide injections with that of 10 U of IL-2. LATI stemming from this association, too, is abolished when mice are irradiated or treated with anti-L3T4 antibody, whereas it is not affected by cyclosporin A or anti-asialo GM1 antibody. Finally, a tumor-specific immune memory is acquired by about 50% of mice after LATI induced by IL-1 beta or 163-171 peptide alone and by about 80% of mice after LATI induced by peptide and lymphocytes from tumor-bearing mice or peptide and IL-2. These findings could lead to the building of a molecularly defined system to induce efficient immune recognition of tumor cells by using a peptide that does not cause any of the several inflammation-associated changes induced by the whole IL-1 beta.     

Interleukin-1 and interleukin-1 fragments as vaccine adjuvants.

The human interleukin-1beta (IL-1beta) domain in position 163-171, comprising the amino acids VQGEESNDK, has been synthesized as a nine-amino-acid-long peptide and used in vivo as a nontoxic HCl salt. The IL-1beta nonapeptide reproduces the immunostimulatory and adjuvant effects of the whole mature IL-1beta, but does not possess any of the IL-1beta inflammatory, vasoactive, tumor-promoting, and systemically toxic effects, nor it can synergize with tumor necrosis factor alpha or other molecules in inducing toxicity and shock. The IL-1beta fragment is active as adjuvant either when administered together with the antigen or if inoculated separately; it can be physically linked to the antigen or used as a discrete peptide. Moreover, the DNA sequence encoding the IL-1beta domain has been included in an experimental DNA vaccine with positive results. Thus, immunostimulatory sequences can be identified within a pleiotropic cytokine like IL-1 and used in the rational design of novel vaccination strategies.     

A short synthetic peptide fragment of human interleukin 1 with immunostimulatory but not inflammatory activity

Short peptide fragments of human and murine interleukin 1 (IL 1) were synthesized on the basis of their predicted exposure on the surface of the molecule in an attempt to identify the minimal structure responsible for the immunostimulatory activity of IL 1. One of these peptides, a fragment of nine residues of human IL 1 beta (VQGEESNDK, fragment 163-171), showed high T cell activation capacity, as judged by its ability to stimulate murine thymocyte proliferation and to potently induce interleukin 2 production in spleen cells. On the other hand, the 163-171 peptide was devoid of prostaglandin-inducing capacity in vitro and pyrogenic activity in vivo, two inflammatory features peculiar to the entire hu IL 1 beta molecule. Thus we propose that this peptide may represent one of the portions of hu IL 1 beta responsible for its immunostimulatory capacity.     

Mechanism of acute toxicity of IL-1 beta in mice

Human recombinant IL-1 beta was able to kill C3H/HeJ mice only when inoculated intravenously at very high doses. IL-1 beta, inoculated at 100 mg/kg i.v. as a bolus, induced a shock-like state characterized by anorexia, severe hypothermia and hypoglycemia and persistent neutrophilia, leading to death in 55% of animals generally between 24 and 48 h. In contrast, the noninflammatory adjuvant IL-1 beta peptide VQGEESNDK (position 163-171) did not induce any toxic effect in vivo, when administered following the same schedule. At variance with what was previously observed in endotoxin induced shock, IL-1 beta induced death was not preceded by appearance of circulating TNF. On the other hand, very high and persistent levels of circulating IL-6 could be detected after lethal IL-1 beta administration. Treatment of mice with ibuprofen or with chlorpromazine, both known to counteract some of the toxic effects of IL-1 in vivo, could protect from IL-1 beta induced mortality. Both drugs, at doses protecting from IL-1 beta induced death, were able to abolish IL-1 beta-induced rise of circulating phospholipase A2 (PLA2) activity, and the subsequent generation of toxic PLA2-derived metabolites.     

Radioprotection by N-palmitoylated nonapeptide of human interleukin-1beta

Interleukin-1beta (IL-1beta) is a cytokine involved in homeostatic processes of the immune system and specifically in inflammatory reactions. The nonapeptide of human IL-1beta (VQGEESNDK, position 163-171) has been shown to retain adjuvant and immunostimulatory activities of the native molecule without any inflammatory and pyrogenic properties. A lipophilic derivative of IL-1beta nonapeptide having a palmitoyl residue at the amino terminus was synthesized in order to determine the effects of such structural modification on its bioactivities. The structurally modified peptide derivative, palmitoylated peptide, significantly protected C3H/HeN mice against potentially lethal doses of ionizing radiation. The dose reduction factor was found to be 1.07. Hematological studies show improved recovery of red blood cells and platelets in irradiated and palmitoylated peptide treated mice as compared with the untreated and irradiated group. These results suggest the importance of the derivatization of small peptides of radioprotective, but toxic cytokines in order to enhance radioprotective activity while reducing unwanted toxic side effects.     

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.     

Structure-function relationship of interleukin-1 giving new insights for its therapeutic potential

The pleiotropic activities of IL-1 have fostered a series of studies on the structure-function relationship in these proteins. In fact, the attempt to dissociate different biological functions of IL-1 should simplify its therapeutic use. About human IL-1, which has been more extensively studied in this respect, enzymatic cleavage of the precursor protein to generate the mature polypeptide appears necessary for its full biological activity. The almost complete integrity of the mature IL-1gb protein is also required for its ability to bind to the receptor and trigger cellular functions. However, by the use of monoclonal antibodies and recombinant or synthetic peptides, it has been possible to map some IL-1gb regions important for different activities. Both N-terminal and C-terminal fragments are important for receptor binding. A domain around amino acids 187-204 is apparently involved in the hyperalgesic effects of IL-1. Finally, the fragment in position 163–171 appears to be responsible for a restricted series of the IL-1 activities, mainly directed to the immune system, although irrelevant for inflammation-related effects and unable of binding to the IL-1 R.It is thus possible, within the sequence of a cytokine, to isolate selectively active domains. This will give us new tools for new therapeutic approaches. Thus, IL-1 might be the prototype of a new generation of cytokines developed with the goal of stimulating specific biological activities without activating the cascade effects which are typical for many cytokines.     

A peptide carrier with a built-in vaccine adjuvant: construction of immunogenic conjugates

A multifunctional carrier combining B/T cell epitopes (i), a built-in vaccine adjuvant (ii), and a universal T cell epitope (iii) for the construction of potent and specific immunogenic conjugates is presented. The IL-1beta(163-171) fragment known to reproduce the immunostimulatory and adjuvant effects of the whole IL-1beta without possessing any of the pro-inflammatory properties of IL-1beta was covalently anchored to the N-terminus of the Sequential Oligopeptide Carrier, SOC(n), formed by the repeating tripeptide unit Lys-Aib-Gly. A promiscuous T cell epitope derived from the tetanus toxin, TT(593-599), was also positioned in the carboxy terminus of SOC(n) as a universal immunogen to provide broad immunogenicity. Selected B/T cell epitopes from the Sm and La/SSB autoantigens, against which is directed the humoral autoimmunity in patients with systemic lupus erythematosus and Sj?gren's Syndrome, respectively, were coupled to the Lys-N(epsilon)H2 groups of the carrier, and the formulated constructs were administered in animals following the conventional immunization protocol of complete/incomplete Freund's adjuvant. The induced immune responses were compared with that produced when the Sm- and La/SSB-reconstituted immunogenic conjugates were injected alone. High titers of specific antibodies recognizing the priming construct, as well as the cognate autoantigen, were obtained when administered alone without the assistance of Freund's adjuvant. It is concluded that our approach provides the conceptual and experimental framework for the development of multifunctional immunogenic conjugates eliciting enhanced, specific, and prolonged humoral response for usage as human vaccine candidates.     

IL-1beta directly excites isolated rat supraoptic neurons via upregulation of the osmosensory cation current

Previous studies have shown that IL-1beta can excite the magnocellular neurosecretory cells (MNCs) of the hypothalamus. However, it is not known whether IL-1beta can have direct IL-1 receptor type 1 (IL-1R1)-mediated effects on MNCs, and little is known about the cellular mechanisms by which IL-1beta influences electrical activity in these cells. Here, we used patch-clamp recordings to examine the effects of IL-1beta on acutely isolated rat MNCs. We found that IL-1beta directly excites MNCs in a dose-dependent manner and that this response can be blocked by an inhibitor of the IL-1R1. Voltage-clamp analysis of the current evoked by IL-1beta revealed a linear current-voltage relationship between -90 and -20 mV, and a reversal potential near -35 mV. This value was not affected by reducing the concentration of chloride ions in the external solution, indicating the involvement of a nonselective cation conductance. The effects of IL-1beta were inhibited by Na-salicylate, an inhibitor of cyclooxygenase. Moreover, the effects of IL-1beta were mimicked and occluded by PGE2, and were inhibited by AH-23848, an antagonist of the PGE2 type 4 (i.e., EP4) receptor. The current evoked by IL-1beta was also abolished by 100 microM gadolinium (Gd3+), but was significantly larger when examined in cells preshrunk by negative pressure applied via the recording pipette. IL-1beta alone did not cause changes in cell volume nor in the mechanosensitivity of MNCs. We conclude that IL-1beta directly excites MNCs via an IL-1R1-mediated induction of PGE2 synthesis and EP4 receptor-dependent autocrine upregulation of the nonselective cation conductance that underlies osmoreception.     

Lack of involvement of lipocortin 1 in dexamethasone suppression of IL-1 release

The annexin lipocortin 1 is reported to mediate some anti-inflammatory effects of glucocorticoids, but the mechanisms of this mediation are incompletely understood. The involvement of lipocortin 1 in glucocorticoid inhibition of monocyte interleukin 1beta (IL-1beta) release has been investigated. Treatment of peripheral blood monocytes with 2 mug/ml lipopolysaccharide potently increased IL-1beta release (p = 0.001) and dexamethasone (10(-7) M) significantly reduced both resting and stimulated IL-1beta release (p = 0.009). A neutralizing monoclonal antibody to lipocortin 1 (0.5-50.0 mug/ml) was unable to inhibit this effect and recombinant lipocortin 1 (2 x 10(-6) M) and 188aa lipocortin 1 fragment (10(-8)-10(-6) M) had no effect. It is concluded that lipocortin 1 is not involved in the inhibition of monocyte IL-1beta release by glucocorticoids.     

A short synthetic peptide fragment of human interleukin-1 beta increases both human and murine natural killer activity

The effect of a short synthetic fragment of human interleukin-1 beta (hu IL-1 beta) on natural killer (NK) activity was examined. Peripheral-blood mononuclear cells (PBMC) from normal donors showed a significant increase in NK activity against K562 leukemia cells after preincubation for 18 h with the IL-1 peptide. A similar augmentation was not observed after culturing the cells in the presence of hu IL-1 beta. The increase in tumor cell lysis could not be ascribed to a cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines. Although the peptide enhanced NK cytotoxicity of PBMC, highly purified large granular lymphocytes were not susceptible to its stimulatory effect. The addition to the cultures of antibodies to human interleukin-2 (hu IL-2) completely blocked the peptide-induced boost of NK cytotoxicity, suggesting that IL-2 is mainly involved in the activation process. The ability of the IL-1 peptide to increase NK activity was further confirmed in vivo in the mouse. Cytotoxicity against YAC-1 lymphoma cells, which was very low in the spleen of untreated BALB/c mice, was in fact significantly increased after a single inoculation of the peptide. These data thus indicate that a short synthetic peptide fragment of hu IL-1 beta is able to increase both human and murine NK activity.     

Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 alpha (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1 beta and promoted TNF and IL-1 beta secretion. Similarly, OX8 Ab (CD8 alpha) stimulated NR8383 cells to secrete TNF, IL-1 beta, and NO. Activation of CD8 beta (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1 beta and stimulated secretion of TNF, but not IL-1 beta. Interestingly, anti-CD8 Abs did not stimulate IFN-gamma or PGE2 production, or phagocytosis by macrophages. OX8 (CD8 alpha)-induced TNF and IL-1 beta production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1 beta, stimulation by CD8 alpha is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1 beta production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 alpha (OX8)-induced TNF and IL-1 beta production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1 beta expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.