Immune Cells and Immunosenescence.

Aging is associated with progressive loss of physiological integrity, leading to impaired physical and mental functions as well as increased morbidity and mortality. With advancing age, the immune system is no longer able to adequately control autoimmunity, infections, or cancer. The abilities of the elderly to slow down undesirable effects of aging may depend on the genetic background, lifestyle, geographic region, and other presently unknown factors. Although most aspects of the immunity are constantly declining in relation to age, some features are retained, while e.g. the ability to produce high levels of cytokines, response to pathogens by increased inflammation, and imbalanced proteolytic activity are found in the elderly, and might eventually cause harm. In this context, it is important to differentiate between the effect of immunosenescence that is contributing to this decline and adaptations of the immune system that can be quickly reversed if necessary.

Lactoferrin Prevents Susceptibility of WEHI 231 Cells to Anti-Ig-Induced Cell Death Promoting Cell Differentiation.

Immature B cells are susceptible to apoptosis due to ligation of surface immunoglobulin receptors. The WEHI 231 cell line represents a useful model to study the mode of action of factors preventing apoptosis. In this work we investigated the protective effects of multi-species lactoferrins in anti-mouse Ig-induced WEHI 231 cell death. Bovine milk-derived lactoferrin (bLF), recombinant human lactoferrin expressed in Chinese hamster ovary cells - rhLF(CHO) or in human endothelial kidney cells - rhLF(HEK), and recombinant mouse lactoferrin expressed in Chinese hamster ovary cells - rmLF(CHO), were used. Goat-anti-mouse Ig antibodies were used to induce cell apoptosis. Survival of WEHI 231 cells in culture was measured using the colorimetric MTT method. Expression of signalling molecules and subunits of interleukin 2 receptor was determined by the RT PCR method. The results showed that anti-mouse Ig antibodies inhibited cell growth in a dose-dependent manner. The lactoferrins alone had no effect on the cell survival. The cells exposed to LFs, prior to anti-Ig treatment, were rescued to a significant degree from cell death. Determination of the signalling molecule expression revealed almost complete suppression of caspase-3 and NF-κB1 by bLF in untreated cells, as well as deep suppression of caspase-3, block of Fas, and 4-fold increase of NF-κB1 in cells incubated with bLF prior to anti-Ig treatment. In addition, differential changes in the expression of interleukin 2 subunits upon bLF treatment were found, indicating a process of cell differentiation. In conclusion, we showed that LF-induced cell differentiation in immature B-cell line WEHI 231 was correlated with partial protection of the cells from anti-Ig-induced cell death.

Lactoferrin accelerates reconstitution of the humoral and cellular immune response during chemotherapy-induced immunosuppression and bone marrow transplant in mice.

Experimental evidence from previous studies supports the conclusion that orally administered lactoferrin (LF) restores the immune response in mice treated with a sublethal dose of cyclophosphamide (CP). The aim of this study was to elucidate potential benefit of LF in mice undergoing chemotherapy with busulfan (BU) and CP, followed by intravenous (i.v.) injection of bone marrow cells. CBA mice were treated orally with busulfan (4 mg/kg) for 4 consecutive days, followed by two daily doses of CP delivered intraperitoneally (i.p.) at a dose of 100 mg/kg and reconstituted next day with i.v. injection of 10(7) syngeneic bone marrow cells. One group of these mice was given LF in drinking water (0.5% solution). After treatment, mice were immunized with ovalbumin (OVA) to subsequently measure delayed type hypersensitivity responsiveness and with sheep red blood cells to determine humoral immunity by evaluation of splenic antibody-forming cells. As expected, both humoral and cellular immune responses of mice that were treated with these chemotherapeutic agents was markedly impaired. Here we report that this impairment was remarkably attenuated by oral administration of LF. Humoral immunity fell to levels that were 66-88% lower than that of untreated animals. Humoral immunity of LF-treated animals was equivalent to that of untreated mice within 1 month. Cellular immune responses were inhibited by chemotherapy treatment to a lesser degree, reaching levels that were approximately 50% lower than those of untreated animals. Again, LF mitigated this decrease, resulting in responses that were only slightly lower than those observed in untreated animals. Furthermore, when mice were given a lethal dose of BU (4 x 25 mg daily doses, i.p.) followed by a bone marrow transplant, LF caused enhanced lympho-, erythro-, and myelopoiesis in the bone marrow and appearance of transforming splenic lymphoblasts, similar to effects caused by administration of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). In summary, our study suggests that LF may be a useful agent to accelerate restoration of immune responsiveness induced by chemotherapy in bone marrow transplant recipients.

New cyclosporin A analogue: synthesis and immunosuppressive activity.

A synthetic analogue of cyclosporine A, in which an unusual amino acid (4R)-N-methyl-4-butenyl-4-methyl-L-threonine (MeBmt) is replaced with L-threonine (Thr), was synthesized by the solid phase method. Its activity in the humoral response to sheep red blood cells in vitro and in vivo in mice was practically the same as that of cyclosporine A used as a standard, whereas the analogue studied exerted a significantly stronger effect in the delayed type hypersensitivity to sheep red blood cells in mice.

Immunologically active nonapeptide fragment of a proline-rich polypeptide from ovine colostrum: amino acid sequence and immunoregulatory properties.

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. To study the relationship between the structure of PRP and its immunomodulatory properties, the polypeptide was digested by chymotrypsin. Products of the proteolysis were separated by gel filtration and three fractions were obtained: PRP-1, PRP-2 and PRP-3. The activity of the fractions was compared with the activity of the untreated PRP. It was found that PRP-1 was inactive, whereas PRP-2 and PRP-3 showed an activity in the regulation of the immune response assayed by measurement of PFC, and by studying effects on delayed hypersensitivity, formation of autologous rosette-forming cell, and sensitivity of thymocytes to hydrocortisone. The activity of PRP-2 and PRP-3 was comparable to the activity of PRP. The PRP-3 fraction of low mol. wt was further purified and a pure nonapeptide of mol. wt 1000 (PRP-3b) was isolated. The amino acid sequence of PRP-3b was: Val--Glu--Ser--Tyr--Val--Pro--Leu--Phe--Pro. The nonapeptide showed the full spectrum of biological activities of PRP. Comparison of terminal amino acid suggested that PRP-3b was neither the NH2- nor the COOH-terminal fragment of PRP. The amino acid sequence of the nonapeptide indicated that PRP-3b is different from other known immunomodulators.

The unusually high immunomodulatory activity of thymopentin analogue Pro5-TP-5.

The immunomodulatory potency of a series of proline-containing thymopentin (TP-5) analogues was investigated by PFC (in vitro and in vivo) test and by GvH reaction. It was found that the substitution of Asp in position 3 of TP-5 by D-Pro yields a distinctly more active compound than that obtained by substitution of Asp3 by L-Pro. Pro5-TP-5 showed very strongly enhanced activity as compared with TP-5; the effect is especially well obvious in PFC in vitro (15-fold enhancement of activity).

Immunoregulatory activity of substance P fragments.

The C-terminal SP7-11 pentapeptide (Phe-Phe-Gly-Leu-Met-NH2) was found to suppress in vitro the immune response in a dose of 1-5 micrograms/ml. It produced also a distinct immunosuppression in vivo, by both per os and intraperitoneal, applications. In contrast, the N-terminal SP1-4 fragment (Arg-Pro-Lys-Pro) suppressed the response at a dose of 0.1 microgram/ml, but stimulated it slightly at higher doses (1-5 micrograms/ml). A structural analog of SP1-4 (Gly-Pro-Arg-Pro tetrapeptide) was found to be a strong immunosuppressor at a dose of 5 micrograms/ml, indicating the importance of N-terminal basic residue for the immunoregulatory activity of intact SP.

Systemic or local co-administration of lactoferrin with sensitizing dose of antigen enhances delayed type hypersensitivity in mice.

Lactoferrin (LF), a major defense protein synthesized and stored in granulocytes has been implicated in maintaining immune homeostasis during an insult-induced metabolic imbalance. In this study, we demonstrated that lactoferrin augments the delayed type hypersensitivity (DTH) response to specific antigens in mice. Lactoferrin (LF) was given to mice orally or intraperitoneally (i.p. ) at the time of immunization, or subcutaneously (s.c.) in a mixture with the immunizing doses of the following antigens, sheep red blood cells (SRBC), Calmette-Guerin bacillus (BCG) or ovalbumin (OVA). A DTH reaction was determined 24 h after administration of an eliciting dose of antigen as a specific increase in foot pad swelling. Lactoferrin enhanced DTH reaction to all studied antigens in a dose-dependent manner. Lactoferrin (LF) given to mice in conjunction with antigen administered in an incomplete Freund's adjuvant induced the DTH response at the level of control mice given antigen in a complete Freund's adjuvant. In addition, LF remarkably increased DTH response to a very small, otherwise non-immunogenic SRBC dose. The increase in DTH response was less pronounced for orally administered LF than for any other routes of administration, however, statistically significant augmentation was demonstrated for each antigen studied. Although the costimulatory action of LF was accompanied by the appearance of bovine lactoferrin-specific cellular responses in mice, it is very unlikely that such responses will be generated in humans, since bovine lactoferrin is a dietary antigen to which a tolerance has been acquired. Considering the involvement of LF in generation of stimulatory signals during the induction phase of an antigen specific immune responses, we suggest that LF may be useful for development of safer and more efficacious vaccination protocols.

Immunostimulatory activity of lactotransferrin and maturation of CD4- CD8- murine thymocytes.

Human milk lactotransferrin at a concentration ranging from 1 to 10 micrograms/ml stimulated up to 5 times the humoral immune response to sheep red blood cells, expressed as the number of plaque-forming cells, when injected into mice 3 h before immunization. Further, lactotransferrin-treated thymocytes given intravenously into mice, enhanced the immune response to sheep red blood cells to the same extent as IL-1. In vitro, studies showed that CD4- CD8- thymocytes incubated with lactotransferrin and added to the splenocyte cultures, increased the immune response to sheep red blood cells. Flow cytometry analysis studies indicated that, after an overnight incubation with human lactotransferrin, CD4- CD8- thymocytes acquired the CD4 antigen characteristic for the helper cell phenotype. Taken together, these results suggest that lactotransferrin stimulates the immune response by a process which involves the promotion of T cell differentiation.

The effect of Ureaplasma diversum activated mononuclear leukocytes on the development and interferon-tau production by bovine IVF-derived embryos.

Ureaplasma diversum is an opportunistic pathogen of the bovine genital tract causing herd outbreaks of granular vulvitis, abortion and infertility. Early embryonic death probably contributes to reduction of the reproductive performance in cows, however, pathogenesis of the disease remains obscure. The aim of the study was to examine whether activation of mononuclear leukocytes by U. diversum may affect embryo development and IFN-tau production. Bovine peripheral blood mononuclear leukocytes were cultured with U.diversum antigen for 24 h. The levels of IL-1, TNF-alpha, NO and GM-CSF in the cell culture supernatants were measured. IVF-derived embryos were cultured in the presence of supernatants from activated leukocytes. The development of embryos until day 6 postinsemination and the rate of morulae/blastocysts were determined. IFN-tau production in supernatants of cultured embryos was examined by inhibition of a virally-induced cytopathic effect. The results showed that U. diversum stimulated mononuclear leukocyte production of IL-1, TNF-alpha and NO. Supernatants from U. diversum-activated cells did not impair the rates of the embryo development and blastocyst formation. The products of activated leukocytes increased the IFN-tau production by cultured blastocysts. This suggest that U. diversum infection provides leukocyte-mediated signals for developing embryos for generation of additional production of cytokine - an important component of innate immunity.

The immunomodulatory activity of tetra- and tripeptides of tuftsin-kentsin group.

It was found that tuftsin (Thr-Lys-Pro-Arg) and its tetrapeptide analogues with the same amino acid composition, but different sequences, demonstrate immunosuppressive activity for the humoral, but not (excepting the Thr-Lys-Arg-Pro tetrapeptide) for the cellular, immune response. The splitting of N-terminal residues from these tetrapeptides leads, however, to formation of tripeptides that are active in both humoral and cellular immune response tests. Thus, the biological degradation of peptides of the type investigated can seriously modulate their immunobiological activities. We also found that tetrapeptides Thr-Arg-Lys-Pro and Thr-Lys-Arg-Pro, as well as tripeptides Arg-Lys-Pro and Lys-Arg-Pro, distinctly differ in their immunomodulatory activities, although the structural differences consist in this case only in the displacement of two basic amino acid residues.

Immunosuppressive activity of antamanide and some of its analogues.

In connection with our discovery of a strong immunosuppressive activity of cyclolinopeptide A (CLA), we investigated immunosuppressive properties of antamanide and a number of its analogues, including symmetrical antamanide, and compared them with the activities of cyclosporin A and CLA. The peptides were investigated by using plaque forming cell (PFC), graft-versus-host (GvH), delayed type hypersensitivity (DTH), and autologous rosette formation cell (ARFC) tests. Antamanide and symmetrical antamanide exhibit an immunosuppressive activity lower than CLA. Linear antamanide fragments are also active. At higher concentrations of the latter peptides, toxic effects occur.

Immunosuppressive activity in the series of cycloamanide peptides from mushrooms.

The immunosuppressor activity of the cycloamanides A, B, C, and D, and two of their D-amino acid residue-containing analogues, was examined using PFC (plaque forming cell) and DTH (delayed type hypersensitivity) tests. It was found that cycloamanide A (CyA A, II) [c-(Phe-Phe-Ala-Gly-Pro-Val-)] and its D-Phe-containing analogue III [c-(Phe-D-Phe-Ala-Gly-Pro-Val-)] are the most potent immunosuppressors of the whole series. The retroanalogue of III [c-(D-Phe-Val-Pro-Gly-Ala-)] was found to be less active than III. The immunosuppressor activity of O-carboxymethyl-Tyr6-antamanide (I) was also tested. It was found that the substitution of one of the Phe residues of ANT by O-carboxymethyl-Tyr does not substantially affect the immunosuppressor activity.

New analogs of the immunoregulatory PRP-pentapeptide.

To determine the role of consecutive amino acid residues in the immunoreactivity of the shortest active fragment of PRP, a series of analogs substituted by L-alanine in successive positions of the peptide chain was synthesized. The immunological investigations were carried out in several models: the immune response to T-cell dependent antigen, SRBC (in vivo and in vitro) and to T-cell independent antigen, PVP in vitro. The immunotropic action of the peptide was, in addition, verified with respect to thymocytes forming autologous rosettes. The results of these experiments revealed immunotropic activity (in all tests) of the analog containing alanine instead of proline in position 5 of PRP-pentapeptide.

Lactoferrin inhibits the effector phase of the delayed type hypersensitivity to sheep erythrocytes and inflammatory reactions to M. bovis (BCG).

Bovine lactoferrin (BLF) given into mice, sensitized to SRBC, together with the eliciting dose of antigen, inhibits very strongly the DTH reaction measured after 24 h by foot pad swelling. Administration of BLF at 48 or 24 h before eliciting the DTH reaction was not effective, however, BLF suppressed the reaction when given at the peak of the inflammatory process. The effects of BLF were strongest when the protein was injected intravenously. Intraperitoneal or intramuscular administrations of BLF were less inhibitory. In addition, BLF diminishes, although to a much lesser degree, the inflammatory reactions induced by BCG. The inhibitory action of BLF does not involve liver since treatment of mice with galactosamine does not reverse the inhibition. Studies on cytokine production revealed that peritoneal macrophages, derived from mice pretreated with LF, have an increased ability to produce in vitro IL-6 after induction with LPS. In addition, we demonstrated that inhibition of macrophage migration, mediated by migration inhibition factor, is abolished by BLF. Lastly, the inhibitory effect of BLF could not be transferred with serum from donors treated with BLF. In summary, the data reveal the inhibitory properties of LF, administered systematically, in relation to locally induced inflammation.

A biorhythm in the humoral immune response to SRBC in mice.

We reveal an existence of a biorhythm in the humoral immune response to SRBC in vivo and in vitro, basing on our experimental data registered in the years 1978-1991. It appears that the immune response to SRBC undergoes regular cycles of high and low responsiveness with 4-week intervals. The biorhythm is not dependent on sex or strain of mice. The importance of the phenomenon in the laboratory practice is discussed.

Interleukin-1 decreases the level of thymocytes forming rosettes with autologous erythrocytes: a new method of determination of interleukin-1 activity.

It was demonstrated that human, recombinant Il-1 significantly inhibited formation of autologous rosettes by thymocytes. In addition, Il-1 increased the percentage of thymocytes resistant to hydrocortisone. Il-2 was not active in both tests. A new method of determination of Il-1 activity, based on the inhibition of the autologous rosette formation by Il-1 is described. The activity of Il-1 in the supernatant from macrophage cultures, stimulated with carbonyl iron powder, was calculated. The lack of Il-2 interference and no necessity to handle radioactive materials, are the advantages of the new method.

Functional maturation of thymocytes: recruitment of helper cells in the humoral immune response to SRBC in vitro by recombinant interleukin 1 (Il-1).

In this report we demonstrated that recombinant human interleukin 1 alpha (Il-1) can directly promote generation of helper cells in the humoral immune response to SRBC in vitro from their intrathymic precursors. Using negative elimination of thymocytes by means of monoclonal antibodies directed against thymocyte antigens L3T4, lyt 2 and CD3 with complement, we showed that Il-1 interacted with L3T4-, lyt 2-, CD3- T cell precursors. We found, in addition, that recombinant Il-1 induced in these cells expression of L3T4, lyt 2, CD3 and T cell receptor (TCR). The results presented in this communication are compatible with the view that Il-1 is responsible for the recruitment of functional T cells in the afferent phase of the immune response.

Modification of immunologic response. IV. A factor in the serum of mice treated with streptomycin, its character and mode of action in vitro.

A factor contained in the serum of mice treated with streptomycin (SM) when added to spleen cell cultures stimulated the primary immunologic humoral response to sheep red blood cells (SRBC) in vitro. The magnitude of the immunologic response was measured as the number of specific antibody-producing cells. By the use of an immunoadsorbent, it was shown that the factor is not SM or a protein-SM complex remaining in trace amounts in the serum of mice treated with this antibiotic. Studies on the mechanism of stimulation by this factor showed that it reacts in a late stage of the immunologic response to already differentiated precursors of antibody-producing cells, and that higher concentrations of this substance added to cultures, instead of stimulating, inhibit producing of antibodies to SRBC. The mechanism of the stimulating action of this factor on antibody-producing cells is discussed.

Cells involved in autologous rosette formation in mice. II. Identification of cells forming autologous rosettes in peripheral blood.

In the study on mice, we showed that autologous rosette forming cells (ARFC) in peripheral blood belong to two categories. First, representing the bulk of ARFC are "null" cells, the other cells are Lyt 123+. The biological significance of the cells which form rosettes in the periphery remains still unknown.