Immunostimulating activities of the novel peptidomimetic L-glutamyl-histamine

Abstract

An original representative of histamine-containing peptidomimetics L-glutamyl-histamine (L-Glu-Hist) was synthesized and characterized as a cytokine mimic leading to cellular responses of improved specificity. The energy-minimized 3-D conformations of L-Glu-Hist derived from its chemical structure resulted in stabilization for Fe²⁺ chelating complexes. L-Glu-Hist accelerated the decrease of ferrous iron in the ferrous sulphate solution in a concentration-dependent mode and showed the ferroxidase-like activity at concentrations less than 3 mm in the phenanthroline assay, whereas in the concentration range 3–20 mm L-Glu-Hist restricted the availability of Fe²⁺ to phenanthroline due to binding of ferrous ions in chelating complexes. L-Glu-Hist showed a stimulatory effect on phosphatidylcholine liposomal peroxidation (LPO) catalysed by the superoxide anion radical ()-generating system (Fe²⁺+ ascorbate) at low (less or about 1 mm) L-Glu-Hist concentrations and both revealed the inhibitory effect on LPO in this system of high (∼ 10 mm) L-Glu-Hist concentration. L-Glu-Hist released in concentrations which stimulated [³H]-thymidine incorporation into DNA and proliferation of mouse spleen lymphocytes and mononuclear cells from human blood. The structural peptide-like analogues of L-Glu-Hist such as L-Glu-Trp, carcinine (β-alanylhistamine), but not L-Pro-Glu-Trp were active in stimulating thymidine incorporation and in inducing proliferation of mononuclear cells compared to mitogen concanavalin A at doses 2·5–25·0 µg/ml. Our data provide evidence that L-Glu-Hist may act as a very fast and sensitive trigger for lymphocyte proliferation and immunoregulation.

Introduction

Reactive species derived from oxygen and nitrogen serve as intracellular messengers that drive signal transduction [1,2]. Oxidation-reduction or redox-dependent reactions have proved to be important in regulating numerous processes that determine the physiological and pathophysiological function of cells and tissues. For example, lymphocytes and human fibroblasts constantly generate small amounts of superoxide radical as growth regulators [2–5]. Superoxide release has also been reported from endothelial cells [6] and smooth muscle cells [7], albeit at levels of one to two orders of magnitude less than that encountered in the case of stimulated macrophages or neutrophils. The engulfed foreign particles in the plasma membrane vesicle are exposed to a high flux of superoxide radicals in the phagocyte cytoplasm which form H₂O₂ via the dismutation reaction and some of the activated oxygen species are also released extracellularly. Once the phagocytic vacuole is formed, fusion with other granules in the neutrophil cytoplasm releases myeloperoxidase, which utilizes H₂O₂ as a co-substrate, with chloride producing the reactive oxidant hypochlorous acid.

Most of the proteins that play key roles in proliferative signal transduction function in a membrane environment or in close association with membranes, and it is established that the activity of integral membrane proteins is modulated by the lipids of the bilayer [8]. Thus protein kinase C has a very specific lipid requirement for its activity, namely phosphatidyl serine [9]. In the presence of lipid hydroperoxides within cell membranes the perturbation of membrane organization occurred due to their polarity. The plasma membrane-bound enzyme Na⁺/K⁺-ATPase important for cell viability can be inactivated by radicals produced during lipid peroxidation (LPO) [10]. However, at low ‘non-toxic’ concentrations of LPO products some effects have been observed which have considerable relevance to cell proliferation. These include the stimulation of adenylate cyclase and phosholipase C activity in cell membranes [11,12], an inhibition of ornithine decarboxylase activity [13] and the expression of early growth regulating genes [14,15]. Consistently, strategies to modulate intracellular redox status by antioxidants and other redox enhancing agents show remarkable therapeutic potential.

Recently the original family of histamine-containing peptidomimetic compounds has been synthesized [16,17]. Its first-representative natural pseudodipeptide carcinine (β-alanylhistamine) has been described as a universal antioxidant in both lipid and aqueous domains with repairing activity to cell membrane damage linked physiologically to L-carnosine (β-alanyl-L-histidine) in the carnosine–histidine–histamine metabolic pathway and endowed with higher resistance to enzymatic hydrolysis comparatively to L-carnosine. Because the receptor systems of histamine are thought to play a central role in growth, wound healing and various types of shock [18–21], as well as having influences on mammalian cardiac, renal, pulmonary, gastric, neurological, ocular and immunological physiology [22–25], we decided to examine the immune cell-related cytokine-like activity of the novel histamine-containing peptidomimetic L-glutamyl-histamine (L-Glu-Hist) using the proliferation test of lymphocytes in vitro. We present evidence in this study that the L-Glu-Hist peptidomimetic compound can both stimulate and inhibit the metal-dependent generation of lipid-derived peroxide intermediates as well as concomitantly release low concentrations of oxygen free radicals and scavenge a number of free radicals such as superoxide () free radicals. The limited release of oxygen free radicals by L-Glu-Hist occurs in concentrations of this peptidomimetic which stimulate proliferation of the cultured immune cells, indicating that this step can be a sensitive trigger for immunomodulation and lymphocyte proliferation.

Materials and methods

Chemicals and biological reagents

All chemicals were of reagent grade; L-carnosine was obtained from Neosystems Laboratories (Paris, France); carcinine 2HCl was provided by Exsymol SAM Laboratories (Monaco, Principaute de Monaco); L-histidine and histamine were obtained from Sigma Chemical Co. (St Louis, MO, USA); superoxide dismutase (SOD) was derived from Serva (Heidelberg, Germany). Synthetic peptides L-Glu-Trp and L-Pro-Glu-Trp were synthesized according to specifications obtained from Dr Y. A. Semiletov (Institute of Virology AMS, Moscow, Russian Federation). All peptides were purified by preparative reverse phase high performance liquid chromatography (HPLC) with a demonstrated level of purity > 98% for peptides.

Analytical data for L-Glu-Hist

L-Glu-Hist was synthesized according to the specifications of Exsymol SAM Laboratories (Fig. 1) [16]. To ascertain the physicochemical characteristics of L-Glu-Hist as a pure compound, NMR spectra were recorded in D₂O on an impulsed Fourier-transformed spectrometer (Bruker AC 200–200 MHz). [¹³C]NMR spectra were recorded in D₂O solution on a Bruker AC 200 spectrometer at 50 MHz. Mass spectra of the compound were obtained on a Finnigan INCOS-500 E mass spectrometer using the solid probe and electron impact at 70 eV, chemical ionization with isobutane at recording temperature 200°C. Analytical thin layer chromatography (TLC) was performed on Silica Gel 60 F254 plates (Merck) using isopropyl alcohol/25% ammonia/H₂O (l4 : 1 : 5) as solvent, by volume. The plates were developed with a solution containing 0·2% ninhydrin, 0·5% cadmium acetate and 2% acetic acid in ethanol. IR spectra were monitored on a Nicolet 5-PC spectrophotometer. Reverse phase analytical HPLC was performed using a Philips PU 4811 chromatography system equipped with a Waters 486 Tuneable Absorbance Detector. Twenty µl of a solution containing 1 mg of the sample dissolved in 20 ml of eluent was injected onto a column (C18 Waters Symmetry® 4·6 × 250 mm) packed with 5 µm silica beads). The column was eluted isocratically at 25°C with 0·1% aqueous solution of trifluoroacetic acid (pH 2·5) over a period of 6 min at a flow rate of 1 ml/min. Eluates were monitored for absorbance at 211 nm. The melting point of the compound was determined on an Electrothermal 9100 melting-point apparatus. The product was collected to give melting point (mp) = 174–177°C, TLC R_f = 0·5, HPLC retention time = 3·5 min, calculated for C₁₀H₁₆N₄O₃ C,50·0; H, 6·7; N, 23·3; found: C,49·75; H,6·89; N, 23·65.

Fig. 1.

Structure of L-glutamyl-histamine, shown as chemical structure.

Molecular modelling

Low-energy 3-D conformations of L-Glu-Hist were derived using the PM₃ method of the mopac 6·0 program [26,27].

Fe²⁺-chelating and ferroxidase-like activity

The ability of L-Glu-Hist to decrease the concentration of free ferrous ions in Tris HC1 buffer (100 mm, pH 7·4) was monitored by the 1,10-o-phenanthroline chelating assay modified from Yoshikawa et al. [28]. The reaction was started by the addition of 12·5 µm FeSO₄ to the reaction mixture which contained 3–20 mm L-Glu-Hist. Sixty min after incubation at 37°C, the reactions were stopped by the addition of 100 µm 1,10-o-phenanthroline (Serva), and A₅₁₅ was read immediately. The ferroxidase activity was also discriminated from the rate of ferrous iron oxidation in the presence of 5 mm ascorbic acid using ethylinediaminetetraacetic acid (EDTA) as a chelating standard. The concentration of (Fe²-1,10-o-phenanthroline) chelating complex was determined using the molar extinction ɛ₅₁₅ = 10931 m⁻¹cm⁻¹.

Superoxide anion radical () generation assay

concentration was determined by the SOD inhibitable cytochrome c reduction assay [29]. Xanthine oxidase (grade I from buttermilk; Sigma, Oxidoreductase, EC 1·1·3·22) (0·02 U/ml) and xanthine-Na-salt (0·025 mm) (Serva) were used to generate in the absence of iron ions (+ 0·1 mm EDTA) or in the presence of iron ions (– EDTA). Reaction mixture (final vol. 2·0 ml) contained 50 µm cytochrome c (Serva), 50 mm KH₂PO₄/KOH buffer, pH 7·4 ± 0·1 mm EDTA, with or without 4·94 U/ml SOD and added the tested compound (L-Glu-Hist). The reaction rate of cytochrome c reduction was monitored spectrophotometrically using the optical density at 550 nm and at 25°C. The concentration of radicals was calculated from the SOD-inhibitable cytochrome c reduced using Δɛ_550nm = 2·1 × 10⁴m^–l × cm⁻¹.

Peroxidation of liposomes

The techniques for phospholipid extraction, purification, preparation of liposomes (reverse-phase evaporation technique) and peroxidation of liposomes during the incubations at 37°C have been described previously [30,31]. The ability of L-Glu-Hist or related compounds (see Table 1) to affect LPO in liposomes was controlled [31].

Table 1.

Effect of imidazole-containing compounds on the iron-ascorbate cata1yzed lipid peroxidation. Peroxidation was initiated by adding 2·5 µm FeSO₄ and 200 µm ascorbate in 0·1 m Tris/HC1 buffer, pH 7·4 to the reaction mixture. Lipid peroxidation products were measured by reaction with TBA (see Materials and methods). Data represent mean of three to five experiments. Oxidation substrate: PC liposomes (1 mg/ml); catalyst Fe²⁺+ ascorbate; time of incubation at 37°C 60 min. Data represent means (%) (n = 3–5).

Testing compound (concentration)	Percentage of control
None	100
Carcinine (10 mm) (β-alanylhistamine)	58
Histamine (10 mm)	163
Histamine (1 µm)	145
L-Carnosine (10 mm) (β-alanyl-L-histidine)	47
Histidine (10 mm)	148
Histidine (10 mm) + EDTA (50 µm)	51
L-Glu-Hist (1·33 mm)	119
L-Glu-Hist (10 mm)	76

Cell studies

Preparation of mouse spleen lymphocytes

Male intact BALb/c mice, approximately 5–6 weeks old, housed in a room with controlled illumination (light/dark 12 : 12) and temperature (23 ± 2°C), were used as spleen donors and processed as described previously [27]. Suspension of the spleen cells was then filtered via the sterilized cotton filter and the medium was added for centrifugation containing 199 + 10% fetal calf serum as the medium. The obtained cells were washed twice during the centrifugation procedure at 100–200 g for 10 min [27]. The cell suspension was dissolved to a concentration of 5 × 10⁶ cells/ml. The isolated cells were cultured in 96-well plates at 37°C in a humidified atmosphere of air CO₂ (95 : 5%) in the growth culture medium RPMI-1640 (Sigma) with 20% fetal calf serum, 0·2 m l-glutamine, 10 mm HEPES buffer (pH 7·35), 5·10⁻⁵m 2-mercaptoethanol and gentamycin (50 µg/ml).

Separation of mononuclcear cells from human blood

Peripheral venous blood was collected by venipuncture from healthy blood donors and introduced into the tubes with heparin (10 U/ml) and processed as described previously [27]. The freshly prepared blood was mixed in equal volumes with 0·9% NaCl. After incubation for 15 min at room temperature, 6–8 ml of the obtained mixture was carefully layered onto the Ficoll-Paque (Pharmacia) 3·0 ml solution with a density gradient of 1·077 g/ml. The mononuclear cells were collected at the interface after centrifugation at 380–400 g for 30 min at room temperature [27].

Proliferation of lymphocytes in vitro

Concanavalin A (Con A) (Sigma, type III containing 15% protein) was used as the mitogen activator of T/B lymphocytes with preferential activation of T lymphocytes. The stimulation of cells was performed by the simultaneous addition of cell suspension (100 µl) and 100 µl Con A solution to the 96-well plates (5·10⁵ cells/well) incubated at 37°C for 72 h in a humidified incubator containing 5% CO₂. Optimal mitogenic concentrations of the control or tested stimuli were determined as essential to cause maximum cell response at preserved cellular viability [27]. The filters were dried at 37°C for 40 min and then replaced into the quartz flasks containing the scintillating liquid (5 ml) (4 g POPOP + 0·1 g PPO per l toluene). The radioactivity of incorporated [³H]-thymidine was detected using beta-spectrometer. Incorporation of [³H]-thymidine into lymphocytes determined by this procedure was shown to be correlated with cell growth [32]. The index of cell stimulation (SI) detectable as a stimulation of [³H]-thymidine incorporation was determined by the ratio:

$$\begin{array}{c}\text{counts per minute in cultures with tested compound}\\ \left(\text{mitogen}\right)/\text{counts per min in control cultures}\\ (\text{addition of}\mathrm{0\cdot 9}\%\text{ NaC1})\end{array}$$

Student's t-test was used to determine the statistical significance.

Results

3-D chemical structuring of L-Glu-Hist

The structural issues considered the energy-minimized 3-D conformations of L-Glu-Hist derived from its chemical structure using stick, ball-and-stick and space-filling models. Due to energy differences determined by molecular mechanics, PM₃ semi-empirical quantum mechanics among different peptidomimetic conformations, a dynamic equilibrium of energetically permissible C-linked and N-linked analogues of rotamers, exists in an aqueous solution. The resulting minimized structure shows that a common characteristic for all the calculated conformations is that the claw-like structure of compound results in a proper stabilization for the metal chelating complexes, such as when the complex of iron : L-Glu-Hist is obtained (Fe²⁺ ion is enveloped tightly into the intraspace of the L-Glu-Hist molecule). The calculated lowest energy conformations for metal-bound rotamers are present at neutral pH and result in any apparent significant adverse steric interactions during changes of pH, temperature or competition with a high-affinity metal chelator (such as EDTA). One (L-Glu) or both (L-Glu + Hist) of the peptidomimetic components provide specificity for metal ion binding, quenching or release of a number of free radicals and binding of hydroperoxide in an imidazole–peroxide adduct [33]. Its structure can be related to histamine and both L-Glu residues for a structure–activity exploration. The structural coincidence of L-Glu-Hist moiety with histamine may have an effect on regulatory proteins in the cell(s) and on DNA/genes involved in normal growth and metabolism.

Effect of L-Glu-Hist on the decrease of ferrous iron

L-Glu-Hist accelerated the decrease of ferrous iron in the ferrous sulphate solution in a concentration-dependent mode of 3–20 mm L-Glu-Hist produced by 10–60 min of incubation (Fig. 2a). The kinetic curves presented in Fig. 2a demonstrate that there is a dose-dependent increase in the rate of ferrous iron disappearance. A ferrous iron chelator 330 µm EDTA showed a complete decrease of the accessible 1,10-o-phenanthroline ferrous ions by the second minute after EDTA addition to the ferrous sulphate solution (Fig. 2, curve 8). The rates of decrease of ferrous iron accessible to 1,10-o-phenanthroline in the presence of L-Glu-Hist are indicative of the interference of iron binding (pure ferrous iron chelating) properties at higher > 5–20 mm L-Glu-Hist concentrations (Fig. 2a, curves 3–5) and the acceleration of auto-oxidation of ferrous iron (ferroxidase-like activity) of L-Glu-Hist at lower than or equal to 3 mm concentrations (Fig. 2a, curves 2 and 1). The reference curves (6, 7) in the presence of EDTA (3 and 33 µm) and curve (1) of autoxidation of ferrous iron are shown in Fig. 2a. The rate of significant decrease of ferrous iron below the autoxidation curve indicates that L-Glu-Hist worked as a ferroxidase compound at lower concentrations (less than 3 mm). Determinations in the presence of 1,10-o-phenanthroline/ascorbate (Fig. 2b) revealed that on the addition of 5 mm, and especially 3 mm L-Glu-Hist, ferrous iron decreased significantly (P < 0·05) by the second minute of the incubation below the auto-oxidation level of ferrous iron, indicating the ferroxidase-like activity of low concentrations of peptidomimetic (curves 1–3). After 10 min incubation with a reductant of Fe³⁺ ions ascorbate (Fig. 2b), a corresponding retention of ferrous iron occurred at the baseline level, showing that ascorbate abolished the acceleration of auto-oxidation of ferrous iron by L-Glu-Hist by 10 min of incubation. Higher concentrations of L-Glu-Hist 10 and 20 mm (Fig. 2b, curves 4 and 5) maintained ferrous iron above the auto-oxidation curve by the second minute. Compared to the high relative affinity of phenanthroline for ferrous ion, this type of kinetics indicates the rising chelating ability of the L-Glu-Hist peptidomimetic to Fe²⁺ in a dose-dependent order of peptidomimetic. When control incubations of the ferrous sulphate solution were performed with 330 µm EDTA (pure chelator), it was found that 1,10-o-phenanthroline-bound ferrous iron increased by the second minute of incubation with ascorbate and recovered further to 25% of the level reached after 10 min during the reduction with ascorbate (Fig. 2b, curve 8).

Fig. 2.

Effect of L-glutamyl-histamine on the decrease of ferrous iron determined by 1,10-o-phenanthroline assay in the presence of 12·5 µm ferrous sulphate (a) or in the presence of 12·5 µm ferrous sulphate + 5 mm ascorbic acid (b). The data points are the means of two independent determinations and are representative of three independent experiments. The standard error of the mean value for each point is ≤3% of the mean value. Details of incubations are presented in the Materials and methods section. (1) Fe²⁺, control incubation; (2–5) Fe²⁺ + l-Glu-Hist (3, 5, 10, and 20 mm, respectively); (6–8) Fe²⁺+ EDTA (3, 33, and 330 µm, respectively). Samples taken at zero time and at the time intervals indicated in (a, b) and were used immediately for measurements.

Action of L-Glu-Hist on superoxide anion radical ()

A mixture of xanthine and xanthine oxidase at pH 7·4 generates , which can be detected by its ability to reduce ferricytochrome c³⁺. Any added compound that is itself able to react with should decrease the rate of reduction of this substance. The tested compound L-Glu-Hist itself does not reduce cytochrome c. Addition of the free metal ions chelator 0·1 mm EDTA to the incubation medium containing reactive compounds significantly diminished the rate of cytochrome c reduction in the xanthine–xanthine oxidase system. L-Glu-Hist had both appreciable inhibitory and stimulating effects on the rate of reduction of cytochrome c by (Table 2). It had an essentially inhibitory effect at high concentrations (10–20 mm) of peptidomimetic. The effects of L-Glu-Hist on reduction of cytochrome c were not connected with inhibition of the xanthine oxidase enzyme itself by L-Glu-Hist because the concentrations used do not effect the conversion of xanthine into hypoxanthine (as judged by the decrease in xanthine), recorded as the change in optical density at 250 nm. EDTA (0·1 mm) clearly does not abolish the inhibitory action of L-Glu-Hist, but acts as an inhibitor itself to the reduction of cytochrome c in much the same way as L-Glu-Hist, because the reduction rate upon addition of EDTA changes more (from 5·6 to 3·9) than the largest effect seen with L-Glu-Hist (5·6–4·6). However, a 0·05 mm concentration of L-Glu-Hist showed effectiveness in the stimulation of formation in the absence or presence of EDTA (Table 2). Due to the ferroxidase-like activity of small (0·05 mm) L-Glu-Hist concentrations, this peptidomimetic can facilitate superoxide free radical release affecting the concentration of catalytically active ferrous ions in reaction:

Table 2.

Reduction of cytochrome c in xanthine–xanthine oxidase (X-XO) system in the presence of L-Glu-Hist.

Conditions	Velocity of reduction of cytochrome c (10−5m−1s−1)
X (0·025 mm) + XO (0·02 U/ml) + cytochrome c (50 µm), pH 7·4
Control	5·6 ± 0·1
Addition of
L-Glu-Hist (0·05 mm)	6·3 ± 0·2*
L-Glu-Hist (10 mm)	5·1 ± 0·1**
L-Glu-Hist (20 mm)	4·6 ± 0·1***
X (0·025 mm) + XO (0·02 U/ml) + EDTA (0·1 mm) + cytochrome c (50 µm), pH 7·4
Control	3·9 ± 0·1
Addition of
L-Glu-Hist (0·05 mm)	6·3 ± 0·2***
L-Glu-Hist (1 mm)	3·9 ± 0·1
L-Glu-Hist (5 mm)	4·2 ± 0·2
L-Glu-Hist (10 mm)	3·0 ± 0·5
L-Glu-Hist (20 mm)	3·3 ± 0·3

Results are the means ± s.e.m. of three to five experiments. Significant differences from controls

^*P < 0·05,

^**P < 0·01,

^***P < 0·001

$${\text{Fe}}^{\mathrm{2+}}+{\text{O}}_2\to {\text{Fe}}^{\mathrm{3+}}+{\text{O}}_{2^{-}}^{.}$$

The superoxide radicals generated in the xanthine oxidase/xanthine system can stimulate the reduction back to the ferrous ions to be catalytic:

$${\text{Fe}}^{\mathrm{3+}}+{\text{O}}_{2^{-}}^{.}\to {\text{Fe}}^{\mathrm{2+}}+{\text{O}}_2$$

Effects of L-Glu-Hist on lipid peroxidation

L-Glu-Hist did not produce any significant effect on peroxidation of PC liposomes in the absence of Fe²⁺ ascorbate catalytic oxidation system. Figure 3a,b presents the kinetic data of MDA and diene conjugates accumulation during peroxidation of PC liposomes, catalysed by the -dependent Fe²⁺-ascorbate oxidation system in the presence and absence of L-Glu-Hist. The results demonstrate that L-Glu-Hist both exhibited a significant stimulatory peroxidative effect for PC liposomes at 210 nm, 42 µm (10 µg/ml) and 1·33 mm concentrations of peptidomimetic and significantly inhibited LPO at 10 mm L-Glu-Hist concentration in the Fe²⁺-ascorbate-catalysed reaction (Fig. 3). These results, together with the above-mentioned data, indicate that L-Glu-Hist has a modest oxidizing activity at low concentrations (less than 1 mm), and works as an antioxidant at high concentrations. The pro-oxidant effect of L-Glu-Hist impurities gives a maximal excess of 2·9 nmol MDA per mg of lipids accumulation over the control incubation in the presence of Fe²⁺ ascorbate by an interval of 20 min, which takes place due most probably to the exhibited during the first minutes of incubation ferroxidase-like activity of L-Glu-Hist more powerful as LPO promoter than free ferrous ion catalysts. The inhibition of TBA-reactive substances (TBARS) accumulation at higher L-Glu-Hist concentrations is due to Fe²⁺ chelation by the peptidomimetic and prevention of radical generation. Figure 3 shows that maximum TBARS and lipid hydroperoxides assessed from the absorbance of diene conjugates reached in the presence of 10 mm L-Glu-Hist at 20 min of incubation decreases at later time-points, which must be due to a loss of existing TBARS or hydroperoxide precursors of MDA and not due to a decreased formation of peroxide compounds. The reduction of lipid hydroperoxides may result from the cleavage of a lipid hydroperoxide with a transition metal complex supplemented with electrons for the reductive reaction LOOH → LOH. Different constituents and imidazole-containing compounds were compared for their ability to inhibit the iron-ascorbate-catalysed LPO (Table 1). The imidazole-containing compounds histamine and histidine exposed peroxidative effects for PC liposomes while carcinine (β-alanylhistamine) and L-carnosine (β-alanyl-L-histidine) showed an ability to inhibit the iron-ascorbate-dependent oxidation of PC liposomes. The addition of 50 µm EDTA significantly inhibited the pro-oxidant activity of 10 mm L-histidine and histamine (Table 1), indicating the role of free iron catalysts. The other tested compounds, i.e. imidazole and β-alanine, were inactive to LPO (see [17]), suggesting that the redox potential of the whole peptidomimetic molecule is essential for exhibition of its pro- or antioxidant activities.

Fig. 3.

Accumulation of lipid peroxidation products (TBARS), measured as MDA (a) and conjugated diene (b) in liposomes (1) (l mg/ml) incubated alone for 60 min and with the addition of the peroxidation-inducing system Fe²⁺+ ascorbate (2). Different concentrations of L-Glu-Hist were added prior to the incubation to the system containing the peroxidation inducers: (3) 210 nm; (4) 42 µm; (5) 1·33 mm; (6) 10 mm. Samples were taken at zero time and at the time intervals indicated (10, 20, 30 and 60 min) and were used immediately for measurement of TBARS (see Materials and methods). A similar amount of sample was partitioned through chloroform and used for detection of conjugated diene dissolved in 2–3 ml of methanol–heptane mixture (5 : 1, v/v). Data represent means ± s.d. (n = 3–5).

Stimulation of lymphocyte proliferation

The estimation of [³H]-thymidine incorporation into DNA is used as a tool for the determination of the proliferation of mouse spleen lymphocytes and immunoregulatory mononuclear cells from human blood in vitro. The pooled data concerning the dose-related effect of L-Glu-Hist and related peptides L-Glu-Trp and L-Pro-Glu-Trp on the incorporation of [³H]-thymidine into DNA of mouse spleen lymphocytes are presented in Table 3. The reference compound mitogen Con A produced a significant stimulating effect on [³H]-thymidine incorporation at doses of 2·5–25 µg/ml with maximum stimulating activity at 10 µg/ml (stimulation index 61·6). As can be seen from the data (Table 3), L-Glu-Hist can specifically regulate proliferation of mouse spleen lymphocytes within the dose range of 2·5–25 µg/ml of L-Glu-Hist peptidomimetic. Within tested low doses of L-Glu-Hist, this compound appeared necessary for cell viability as judged by trypan blue staining. Cell cultures exposed to L-Glu-Hist in the dose range indicated revealed no cell damage nor an obvious loss of cellular density. A dose of 10 µg/ml L-Glu-Hist significantly stimulated the [³H]-thymidine incorporation into the DNA of mouse spleen lymphocytes (stimulation index 42·2). The related L-Glu-containing dipeptide L-Glu-Trp showed significant stimulation of [³H]-thymidine incorporation into the DNA of mouse spleen lymphocytes in vitro at doses of 2·5–10·0 µg/ml, whereas L-Pro-Glu-Trp tripeptide exhibited no stimulatory activity on thymidine incorporation into the mouse spleen lymphocytes in the range of doses studied in the same test. Despite the fact that the structure of L-Glu-Hist is related to histamine, it appears doubtful that the stimulating effects on the proliferation of lymphocytes/monocytes observed with L-Glu-Hist are related to pure histamine residue, but rather to the introduction of L-Glu residue into the peptidomimetic molecule, as the dipeptide L-Glu-Trp has virtually the same effect (stimulating index 37·2) as L-Glu-Hist (stimulating index 42·8; standard deviation: about 50% of the mean value). In the human blood mononuclear cells L-Glu-Hist and mitogen Con A applied at a similar dose range did not change cell viability negatively and showed significant stimulating activity on the proliferation of lymphocytes in the range of 2·5–10 µg/ml doses, with a maximum stimulating effect on the [³H]-thymidine uptake at 5 µg/ml (see Table 4). A histamine derivative of L-Glu-Hist, carcinine, 2HCl β-alanylhistamine showed similarly good biocompatibility and exposed induction of proliferation of mononuclear cells at doses of 5·0–25·0 µg/ml, when it was used alone and in admixture with Con A (Table 4). In both types of cells L-Glu-Hist stimulated [³H]-thymidine incorporation into the DNA and lymphocyte proliferation in our assay system at a low concentration (ranged about 42 µm) of peptidomimetic, coinciding with the finding that this compound also appears to have a pro-oxidant activity to lipid membranes in the Fe²⁺-ascorbate system, interlaced with the release of low concentrations of oxygen free radicals due to the ferroxidase-like activity of L-Glu-Hist. This finding provides a possible mechanism by which L-Glu-Hist can act as a very fast specific and sensitive trigger for lymphocyte proliferation and immunoregulation. A number of different mechanisms may contribute to the mitogen-like activity of the matched tested compounds: Con A, L-Glu-Trp, L-Pro-Glu-Trp and carcinine (β-alanylhistamine).

Table 3.

The effect of L-glutamyl-histamine, related peptides L-Glu-Trp, Pro-Glu-Trp and Con A on [³H]-thymidine incorporation into DNA of mouse spleen lymphocytes in vitro.

Compounds	n	c.p.m./5 × 105 cells ± s.e.m.	Stimulation index	P <
Control (free of mitogen or testing compound)	7	796 ± 276
Con A µg/ml
2·5	5	6620 ±720	8·3	0·001*
5·0	5	26607 ±6689	33·4	0·01*
10·0	4	49002 ±13003	61·6	0·01*
25·0	5	5055 ±1167	6·4	0·01*
L-Glu-Hist µg/ml
2·5	4	6222 ±2608	7.8	0·1
5·0	5	34042 ±17765	42.8	0·1
10·0	6	33589 ±10336	42.2	0·05*
25·0	4	4528 ±3293	5.7	n.s.
L-Glu-Trp µg/ml
2·5	5	3184 ±346	4·0	0·001*
5·0	4	29634 ±8350	37·2	0·01*
10·0	5	13636 ±3501	17·1	0·01*
25·0	5	1000 ±225	1·3	n.s.
L-Pro-Glu-Trp µg/ml
2·5	6	2070 ±852	2·6	n.s.
5·0	5	2318 ±1075	2·9	n.s.
10·0	4	4935 ±3076	6·2	n.s.
25·0	5	3741 ±2405	4·7	n.s.

n = number of determinations;

^*significant differences with control; n.s.: non significant difference with control. Results are the means ± s.e.m. of three to five experiments. The data do not indicate any specific effect of L-Glu-Hist versus L-Glu-Trp, but indicate the input and important role of L-Glu residue in the structure of peptidomimetic in the signalling activity.

Table 4.

The effect of L-glutamyl-histamine, carcinine 2HCl (β-alanylhistamine) and Con A on the proliferation of mononuclear cells (lymphocytes) obtained from blood of the normal human donor, 48 years (in vitro studies).

Compounds	n	c.p.m./5 × 105 cells ± s.e.m.	Stimulation index	P <
Control (addition 6 of RPMI-1640  medium)		1201 ±120
Con A µg/ml
2·5	4	5785 ±320	4·8	0·001*
5·0	3	35544 ±5400	29·6	0·001*
10·0	4	25369 ±2480	21·1	0·001*
25·0	4	872 ±120	0·7	n.s.
L-Glu-Hist µg/ml
2·5	4	9884 ±986	8·2	0·001*
5·0	4	47816 ±1580	39·8	0·001*
10·0	3	28329 ±1520	23·5	0·001*
25·0	5	3039 ±1240	2·5	n.s.
Carcinine
2HCl β-alanylhistamine µg/ml
2·5	4	5765 ±1980	4·8	0·1
5·0	5	18856 ±6800	15·7	0·05*
10·0	4	20297 ±6820	16·9	0·05*
25·0	4	22339 ±4470	18·6	0·001*
50·0	3	15372 ±3888	12·8	0·1
100·0	4	1922 ±420	1·6	n.s.
Carcinine 2HCl β-alanylhistamine (10 µg/ml) + Con A (10 µg/ml)
	3	30145 ±5500	25·1	0·001*

n = number of determinations;

^*significant differences with control; n.s.: non-significant difference with control. Results are the means ± s.e.m. of three to five experiments.

Discussion

A histamine-containing peptidomimetic (L-Glu-Hist) was synthesized and characterized in this study and its role as an immune cells cytokine mimetic leading to cellular lymphocytes proliferation responses was revealed. The L-Glu-Hist peptidomimetic showed stabilization in the formation of ferrous ions chelating complexes and accelerated the decrease of ferrous iron ions concentration in the aqueous medium, demonstrating the ferroxidase-like activity at less than or equal to 3 mm concentrations of L-Glu-Hist peptidomimetic. L-Glu-Hist demonstrated pure ferrous iron chelating properties at higher, >5–20 mm, L-Glu-Hist concentrations responsible for its antioxidant activity in lipid membranes in this concentration range. L-Glu-Hist exhibited a stimulatory effect for PC liposomes peroxidation catalysed by the superoxide anion radical generating system at 210 nm, 42 µm (10 µg/ml) and 1·33 mm concentrations of peptidomimetic.

L-Glu-Hist stimulated DNA synthesis and the proliferation of mouse splenocytes and human peripheral blood lymphocytes. Structural analogues of L-Glu-Hist [L-Glu-Trp, carcinine (β-alanyl-histamine)] were also active in stimulating thymidine incorporation compared to that mediated by Con A. The data provided suggest that L-Glu-Hist can act rapidly as a trigger for lymphocyte proliferation and immune regulation. The study analyses the mechanisms of biological activity of L-Glu-Hist, including its modulating effects on free radical-induced oxidation and function of specific oxygen radicals and radical-derived species as well as its role as a chemical ‘messenger’ to lymphocyte reactivity and spleen cells functions when added directly to the cell culture medium.

In comparison with ‘free’ ferrous ion, the addition of L-Glu-Hist at high concentrations results in (1) decreased peroxidation of lipids, (2) decreased cytochrome c reduction by superoxide anion radicals and (3) decreased lymphocyte proliferation. The magnitude of these effects, however, is modest. These results are attributed to the antioxidant properties of the iron–Glu-Hist complex. On the other hand, our major findings were that at low concentrations, addition of L-Glu-Hist resulted in (1) a modest increase of LPO, (2) a modest increase in the rate of cytochrome c reduction and (3) a strikingly significant increase of lymphocyte proliferation. These results are attributed to the ferroxidase-like activity of the complex at low concentrations.

Although a number of mechanisms may contribute to the underlying processes that give rise to the results observed, including the release of cytokines by activated lymphocytes capable of stimulating several distinct processes, the expression of immunomodulatory proteins and the synthesis and release of hydrophilic glycosaminoglycans, a direct effect of oxygen free radicals released by L-Glu-Hist upon the lymphocyte proliferation, is one possible explanation. It is now clear that superoxide and H₂O₂ can stimulate growth responses in a variety of mammalian cell types when added exogenously to the culture medium. Our results show that the proliferative response to L-Glu-Hist occurs in immune cells cytokines in a dose-dependent manner (10–50 µm), although maximal stimulation of LPO in the -generating system (Fe²⁺+ ascorbate) occurred at different (higher: ∼1 mm) L-Glu-Hist concentrations studied. Free radical release may occur in these instances at a rate insufficient to damage cells, but sufficient to stimulate their proliferation. Because the previous [34,35] and present experiments used fetal calf serum in the incubations, these cells would accumulate a sizeable amount of iron [36] which, upon the addition of L-Glu-Hist, could induce or inhibit free radical release and LPO.

L-Glu-Hist has maximum mitogenic activity on lymphocytes at about 10 µg/ml (corresponds to about 40 µm). This level of L-Glu-Hist was shown to have a modest oxidizing activity and the increase in mitogenic activity is higher than expected from the modest oxidizing activity of L-Glu-Hist. The specificity of mitogenic activity of L-Glu-Hist on lymphocytes can also be related to the L-Glu and Hist moieties of peptidomimetic. This effect cannot be related solely to the oxidative nature of the molecule. The structural derivatives of L-Glu-Hist, such as L-Glu-Trp and carcinine (β-alanylhistamine), but not L-Pro-Glu-Trp, were shown in this study to able to induce lymphocyte proliferation in a dose-dependent manner similar to L-Glu-Hist peptidomimetic. A combination of carcinine and Con A did not exhibit an additive effect. This experiment means that the immune cellular signalling does not necessarily indicate a combination of more specified cellular factors.

Because phenanthroline is added in excess of Fe²⁺, a high binding constant in comparison with L-Glu-Hist would mask the true chelating ability of the peptidomimetic. Analysis of the binding kinetics in the presence of ascorbate could help to distinguish between chelation and oxidation of ferrous to ferric ion as the main factor in the reduced availability of free ferrous ion. The kinetic results provide more support for the statement that reduced availability of ferrous ion is due to chelation at high concentrations of L-Glu-Hist, but to ferroxidase activity at low concentrations of peptidomimetic.

Chemically, the antioxidant activity of L-Glu-Hist shown at high concentrations of peptidomimetic is not due solely to the imidazole moieties of the molecule, as imidazole itself did not show antioxidant activity, and histamine exhibited a pro-oxidant action dependent on the concentration of catalytically active free iron ions (this study and [17]). The antioxidant activity of L-Glu-Hist may involve the reduction of oxidative potential or stabilization of the imidazole radical, due probably to the peptide bond. The comparative lack of the carboxyl group of histamine to histidine is essential for the demonstration of more pro-oxidant than antioxidant activity of the molecule. Because L-Glu-Hist has been shown to be an effective chelating agent for ferrous ions, the ligand acts as an antioxidant at high concentrations, but at low concentrations the peptidomimetic acts as a generator of the reactive oxygen species that it presumably reduces at high concentrations.

Reactive oxygen species include the superoxide molecule (), the hydroxyl radical (HO^•), singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂). The current results do not distinguish whether the reactive oxygen species generated are superoxide, hydrogen peroxide or hydroxyl radical. In aqueous solution two molecules of superoxide can disproportionate into O₂ and hydrogen peroxide, which in turn can generate hydroxyl radical by Fenton-type chemistry with ferrous ion. The exact mechanism and the contribution of , H₂O₂, HO^• and LPO products to lymphocyte stimulation and proliferation is undetermined. Preliminary investigations suggest that stimulation of cyclo-oxygenase by peroxides in the prostaglandin cascade may be important [37]. As an alternative, the signalling of proliferation responses in the immune cells involving released superoxide or hydrogen peroxide may be mediated through the oxidative inactivation of serum protease inhibitors, allowing serum proteases to remodel the cell surface, or glycocalyx, and thereby facilitate or modulate the action of L-Glu-Hist and thus retain the necessary all-important growth specificity [38]. Using the cytochrome c reduction test we have shown that superoxide radicals are at least abundant to stimulate lymphocyte proliferation in vitro, being formed spontaneously due to the ferroxidase-like activity of L-Glu-Hist applied at less than or about 50 µm concentration.

The presence of lipid hydroperoxides within cell membranes will perturb membrane organization due to their polarity. However, it has been found recently that LPO has a dual effect on lipid order [39]. A more ordered or disordered state may result depending on the degree of oxidation and the state of lipid order prior to oxidation. Although an adequate explanation is not yet available for the endogenous LPO encountered in cultured mammalian cells, it is clear nevertheless that the level can vary considerably. Lipid peroxides can yield oxidative breakdown products such as the hydroxyalkenals through non-enzymic pathways. The aldehyde products of LPO, in particular 4-hydroxynonenal (HNE), can react with thiol and amino groups of proteins affecting several cellular activities [40,41]. Such effects occur at HNE (aldehyde) concentrations greater than 10 µm. At concentrations of less than 3 mm L-Glu-Hist showed lower ‘non-toxic’ stimulating activity for TBARS accumulation in the -generating system Fe²⁺+ ascorbate. It is hypothesized that L-Glu-Hist may play a central role in the ‘down-regulation’ of cell proliferation via the production of LPO products at a ‘steady-state’ level which can be catabolized rapidly by normal cells [42].

One of the mechanisms by which antioxidants can protect their biological targets from oxidative stress is the chelation of transition metals such as copper and iron, thus preventing them from participating with peroxides in the deleterious Fenton reaction. At concentrations of 5–20 mm L-Glu-Hist was shown to be an efficient ferrous iron chelating agent which can possess low SOD-like activity in vitro. The iron–L-Glu-Hist complex was sufficient to reduce the superoxide radicals only when EDTA was omitted from the incubation medium. The iron chelating complexes of L-Glu-Hist may serve as scavengers of the superoxide radicals rather than as catalysts. Another antioxidant protection possessed by L-Glu-Hist is that at a 10 mm concentration this compound removes lipid-derived hydroperoxide intermediates catalysed typically by glutathione-requiring enzymes (Se-dependent GSH peroxidases and certain Se-independent enzymes, such as GSH-S-transferase B) [43]. The reduction of lipid peroxides by L-Glu-Hist at high concentrations was monitored experimentally by the diene conjugates measuring test, and tested by the decrease of TBARS during LPO.

The stimulating activity of L-Glu-Hist at low concentrations is more interesting. Our study describes most promising results, as the peptidomimetic is shown to have very similar activity to Con A. In the phenanthroline strong chelating assay the excess FeSO₄ at a concentration of 12·5 µm was added to the reaction mixture and at the concentrations of peptidomimetic that cell stimulation is seen no information on binding and/or presumed ferroxidase activity is presented (Fig. 2). However, the effects indicated, related with the antioxidant or pro-oxidant properties of L-Glu-Hist, are seen in the liposome peroxidation experiments (Fig. 3). The discrepancies between ≤1 mm and 10 mm concentrations of L-Glu-Hist in their action to the iron-catalysed LPO are consistent with the metabolic effects of this peptidomimetic on cellular activities and their roles in metabolism of lipid-derived peroxide intermediates.

In summary, the findings obtained suggest a theoretical avenue through which oxidative damage, acting either directly or as an end-product of cytokine release, may contribute to lymphocyte stimulation. Free radicals perform many critical functions in our bodies in controlling the flow of blood through our arteries, to fighting infection, to keeping our brains alert and in focus. Similar to antioxidants, some free radicals at low levels are signalling molecules − i.e. they are responsible for turning genes on and off. The phenomena outlined in this paper introduce a novel synthetic peptidomimetic, L-Glu-Hist, as a potent immunotherapeutic signalling agent and a tool with an oxygen radicals modulating activity and a specific role as a physiological agonist, leading to the various above-discussed cellular responses, such as lymphocyte proliferation by modulating DNA synthesis.