Suppressive effect of iron on in vitro lymphocyte function: formation of iron polymers as a possible explanation

The evidence presented here indicates that ferric citrate inhibits both the phytohemagglutinin-induced lymphocyte proliferation and the formation of E rosettes by T lymphocytes. These inhibitory effects are only observed in the presence of ferric citrate with a metal to ligand molar ratio of (1:1) but not with ferric citrate (1:20) or sodium citrate. Since ferric citrate (1:1) at a physiological pH tends to hydrolyze and polymerize, we suggest that the inhibitory effect is mediated by the formation of iron polymers.     

Iron exerts a specific inhibitory effect on CD2 expression of human PBL

The effect of in vitro exposure to ferric citrate (Fe-citrate) on the expression of human lymphocyte surface markers was studied. The following markers were examined: E-rosette formation, CD2, CD3, CD4, CD8, CD1, CD22, CD10 and HLA-DR. Pretreatment of peripheral blood lymphocytes (PBL) with Fe-citrate at concentrations ranging from 10(-2) M to 10(-5) M resulted in the exclusive inhibition of E-rosette formation and CD2 expression. None of the other surface antigens examined appeared to be sensitive to the Fe-citrate treatment. Competition experiments further indicated that iron interacts specifically with CD2 on T lymphocytes.     

Human milk contains proteins that stimulate and suppress T lymphocyte proliferation.

The modulatory effect of human milk proteins from colostrum and late milk on the proliferative response of human T lymphocytes activated by mitogens (OKT3 and leucoagglutinin from Phaseolus vulgaris) and alloantigens was studied. High concentrations (10-100 micrograms/ml) of crude colostral milk proteins had an inhibitory effect on T cell growth while low concentrations (0.1-1 microgram/ml) enhanced T cells growth. In contrast, proteins from late milk did not inhibit T lymphocyte proliferation while the enhancing effect was retained. Colostrum was fractionated by ammonium sulphate precipitation and gel filtration on sepharose 6B. The inhibitory activity was recovered in a protein fraction containing lactoferrin as its major component. Lactoferrin was, however, not responsible for the observed inhibition. On the contrary, lactoferrin in most cases augmented the proliferative response induced by polyclonal activators. The inhibitory activity was found to bind concanavalin A-sepharose suggesting an association with glycoprotein. Inhibitory fractions contained glycoproteins of the following molecular sizes 26, 74/76 (doublet), 84, 145 and 160 kD under reducing conditions. The inhibitory effect appeared to be lymphocyte specific since the active fraction did not inhibit the growth of tissue culture cells (HeLa cells and human fibroblasts) or bacteria. Furthermore, the fraction was not toxic for lymphocytes. The inhibitory colostrum factor may prevent the newborn from overreacting immunologically against the environmental antigens encountered at birth.     

Effect of Hepatic Isoferrttins from Iron Overloaded Rats on Lymphocyte Proldzerative Response: Role of Ferritin Iron Content

Iron and ferritin impair a variety of immunological functions. To evaluate the effect of ferritin iron content on rat lymphocyte proliferative response, isoferritins that differ in their iron content and isoelectric point (pI) were isolated from iron overload rat livers by ultracentrifugation (isoferritins with high iron content and low pI) or crystallization (isoferritins with low iron content and high pI) methods. Additionally, commercial horse splenic ferritin (with a lower pI and higher iron content than rat isoferritins) was also tested. Proliferative response to Con A was decreased in a dose-dependent manner in all assays in which spleen cells were incubated with rat and horse isoferritins. However, isoferritins with higher iron contents (rat isoferritin obtained by ultracentrifugation and horse ferritin) caused a greater decrease of proliferative response at 5 and 25 μ/ml than the others. Rat and horse apoferritins showed no inhibitory effect on lymphocyte proliferative response, suggesting that the effect is due to iron probably through the damaging effect of reactive oxygen species generated by iron released by the isoferritins on lymphocyte functions.

Additionally, the role of serum ferritin level on proliferative response was studied in an experimental model of iron overload in rats. An inverse relationship between the proliferative response and serum ferritin levels was observed.

Our results suggest that the inhibitory effect of the isoferritins on lymphocyte proliferative response is due, at least partially, to the iron content of this protein and not exclusively to variation in pi as suggested by other authors. These results are in agreement with the possible immunosuppressor role of ferritin in vivo.     

Effect of Hepatic Isoferrttins from Iron Overloaded Rats on Lymphocyte Proldzerative Response: Role of Ferritin Iron Content

Abstract

Iron and ferritin impair a variety of immunological functions. To evaluate the effect of ferritin iron content on rat lymphocyte proliferative response, isoferritins that differ in their iron content and isoelectric point (pI) were isolated from iron overload rat livers by ultracentrifugation (isoferritins with high iron content and low pI) or crystallization (isoferritins with low iron content and high pI) methods. Additionally, commercial horse splenic ferritin (with a lower pI and higher iron content than rat isoferritins) was also tested. Proliferative response to Con A was decreased in a dose-dependent manner in all assays in which spleen cells were incubated with rat and horse isoferritins. However, isoferritins with higher iron contents (rat isoferritin obtained by ultracentrifugation and horse ferritin) caused a greater decrease of proliferative response at 5 and 25 μ/ml than the others. Rat and horse apoferritins showed no inhibitory effect on lymphocyte proliferative response, suggesting that the effect is due to iron probably through the damaging effect of reactive oxygen species generated by iron released by the isoferritins on lymphocyte functions.

Additionally, the role of serum ferritin level on proliferative response was studied in an experimental model of iron overload in rats. An inverse relationship between the proliferative response and serum ferritin levels was observed.

Our results suggest that the inhibitory effect of the isoferritins on lymphocyte proliferative response is due, at least partially, to the iron content of this protein and not exclusively to variation in pi as suggested by other authors. These results are in agreement with the possible immunosuppressor role of ferritin in vivo.     

Hepcidin messenger RNA expression in human lymphocytes

Hepcidin regulates intracellular iron levels by interacting with and promoting the degradation of ferroportin, a membrane protein and the only known cellular iron exporter. Studies of hepcidin expression and regulation have focused on its effects in innate immunity and as a regulator of systemic iron metabolism. In the present study we characterized the expression of hepcidin messenger RNA (mRNA) in human peripheral blood mononuclear cells (PBMCs) with a focus on peripheral blood lymphocytes (PBLs). We found that (1) all human PBMCs analyzed express basal hepcidin mRNA levels; (2) hepcidin mRNA expression increases after T‐lymphocyte activation; (3) expression by PBLs increases in response to challenge by holotransferrin (Fe‐TF) and by ferric citrate in vitro; (4) the Fe‐TF‐mediated up‐regulation of hepcidin decreases ferroportin expression at the cytoplasmic membrane of PBLs; and (5) silencing of tumour necrosis factor‐α (TNF‐α) abrogates the effect of Fe‐TF. In summary, we show that hepcidin expression determines intracellular iron levels by regulating the expression of ferroportin, as described in other cells, and that inappropriately low expression of hepcidin impairs normal lymphocyte proliferation. The results establish hepcidin as a new player in lymphocyte biology.     

Staphylococcal enterotoxin B as a potent suppressant of T lymphocytes: trace levels suppress T lymphocyte proliferative responses.

Staphylococcal enterotoxins have long been known to be powerful stimulators of T lymphocytes in mouse and man. In a previous study we showed that high concentrations of staphylococcal enterotoxin serotype B (SEB) failed to stimulate strong proliferative responses by Lewis rat T lymphocytes. Moreover, concentrations of SEB (10-50 micrograms/ml) that stimulated optimal mouse T lymphocyte proliferative responses suppressed a mitogen- or antigen-induced rat T lymphocytes proliferative responses. The present study shows that SEB at low concentrations (as low as 10(-3)-10(-4) micrograms/ml) and often also trace levels (about 10(-6)-10(-7) micrograms/ml) suppresses both rat and mouse T lymphocytes proliferative responses to mitogen or antigen. Furthermore, under different circumstances, SEB may have conflicting effects on the same T cells. While high concentrations (1-50 micrograms/ml) of SEB stimulate certain mouse T cell clones, low concentrations or trace levels have a potent suppressive effect on the same clones. The results indicate that the in vitro conflicting effects of SEB on the same T cells are concentration dependent and may reflect its in vivo effects on SEB-reactive T lymphocytes. The suppression of the mitogen- or antigen-induced stimulation of T cell clones by SEB was direct and did not require the agency of suppressor cells. Furthermore, the suppression by low amounts of SEB was not major histocompatibility complex restricted and affected a large proportion of both rat and mouse T lymphocyte subpopulation, regardless of their antigenic specificity. The concomitant suppressogenic and stimulatory characteristics of SEB support the conclusion that, under different conditions, SEB can be considered a "super-suppressogen" as well as a "super-antigen". Overall, the results suggest that SEB, and possibly other bacterial toxins, could be useful in immunomodulation of specific T cell responses.     

CD69 expression reliably predicts the anti-CD3-induced proliferative response of lymphocytes from human immunodeficiency virus type 1-infected patients.

Published studies suggest that mitogenic responses of lymphocytes can be reliably assessed by monitoring the expression of lymphocyte surface CD69 after 24 h of culture with the stimulant. We tested this hypothesis by determining the ability of lymphocyte CD69 expression to predict the outcome (normal or abnormal) of lymphocyte proliferative responses to anti-CD3 in a group of human immunodeficiency virus type 1 (HIV-1)-infected patients (n = 47). Cutoff values for defining normal and abnormal CD69 expression and proliferative ([3H]thymidine incorporation) responses were established with lymphocytes from healthy uninfected controls (n = 20). Lymphocytes from 29 HIV-infected patients exhibited an abnormal proliferative response, and those from 25 of the 29 also exhibited abnormal CD69 expression (sensitivity, 86.2%). Similarly, lymphocytes from 18 HIV-infected patients exhibited a normal proliferative response, and those from 16 of the 18 also exhibited normal CD69 expression (specificity, 88.9%). The predictive value of a normal CD69 result was 80%, and the predictive value of an abnormal CD69 result was 92.6%. These findings demonstrate that HIV-1-associated impairments in lymphocyte activation can be reliably detected by the rapid and nonradioactive CD69 expression assay.     

Somatomedin production and response to mitogen by lymphocytes in children with growth hormone deficiency

Studies on the lymphocyte proliferative activity of the sera from growth hormone (GH) deficient patients have resulted in contradictory observations. The ability of lymphocytes to synthesize somatomedin-C (Sm-C) and thus account for a normal proliferative activity (previously observed by us) of the GH-deficient sera was studied, by measuring Sm-C concentration in the culture medium using a standard radioimmunoassay for Sm-C. The response of lymphocytes to the mitogen phytohemagglutinin (PHA), as determined by the incorporation of 3H thymidine into DNA, was also studied. The Sm-C concentrations in the cultures reflected the Sm-C concentrations of the respective serum added and did not alter with significant increases in the cell number induced by PHA. The lymphocytes from GH-deficient children and normal children were indistinguishable in their ability to respond to PHA. We conclude that lymphocyte proliferation in short-term culture, was not associated with an increase in Sm-C and that in the lymphocyte proliferation assay the sera and the lymphocytes from GH-deficient children respond similarly to the sera and lymphocytes from normal children.     

Inhibitory and stimulatory effects of Pseudomonas aeruginosa pyocyanine on human T and B lymphocytes and human monocytes.

Pyocyanine, a pigment produced by Pseudomonas aeruginosa, has dual dose-dependent stimulatory as well as inhibitory effects on immune responses in vitro as measured by DNA synthesis of human T and B lymphocytes, interleukin-2 (IL-2) production by human T lymphocytes, immunoglobulin production by human B lymphocytes, and monokine production by human monocytes. In general, stimulatory activity was found at low concentrations of pyocyanine, whereas high concentrations of the pigment resulted in an inhibition of responses. At a pyocyanine concentration of 0.1 micrograms/ml or less the proliferation of T and B lymphocytes was enhanced, but at 0.5 micrograms/ml it was suppressed. IL-2 production by T lymphocytes was enhanced at concentrations up to 0.5 micrograms/ml but totally inhibited at 1.0 micrograms/ml. The differentiation of B lymphocytes to become immunoglobulin-producing cells was also enhanced in the presence of low doses of pyocyanine, whereas secretion of immunoglobulin by B lymphocytes was suppressed at all concentrations of pyocyanine. In contrast to the dual effects of pyocyanine on lymphocyte response, lipopolysaccharide-induced IL-1 and tumor necrosis factor release by monocytes was markedly enhanced by low as well as high concentrations of pyocyanine. From these results we conclude that this property of pyocyanine may lead to suppression of specific defense mechanisms and enhance harmful inflammatory reactions of the host during infection with Pseudomonas aeruginosa.     

Lymphocyte stimulation in vitro by orosomucoid glycoprotein

Mitogenesis of human peripheral blood lymphocytes as measured by the uptake of [3H]thymidine was stimulated in vitro by pure orosomucoid glycoprotein when used at concentrations that are considerably lower than the physiological plasma level. The lymphocyte cultures stimulated with PHA or PWM were not affected by low concentration (67 micrograms/ml), but they were mildly suppressed by high concentration (1 mg/ml) of this glycoprotein. The stimulatory response was relatively greater with fractionated T cells than the non-T cells (B cells and monocytes). At 50 micrograms/ml concentration of orosomucoid, the lymphocyte activation was found in randomly selected blood donors which included normal healthy volunteers and patients with T cell immunodeficiency or Alzheimer's disease, demonstrating a consistent immunostimulatory action of this glycoprotein.     

Effect of cyclic adenosine monophosphate on the in vitro DNA synthesis in lymphocytes of patients with chronic lymphocytic leukemia

Deoxyribonucleic acid (DNA) synthesis was investigated in vitro in T and B lymphocyte populations isolated from peripheral blood. The synthesis was determined by measurements of 3H-thymidine incorporation. Only T lymphocytes isolated from peripheral blood of healthy subjects as well as patients with chronic lymphocytic leukemia (cll) showed proliferative ability after stimulation with phytohemagglutinin (PHA). DNA synthesis rate in normal T lymphocytes was enhanced by exogenous cyclic adenosine monophosphate (cAMP) added to lymphocyte culture together with PHA in concentrations of 10(-5) and 10(-4)M. Higher concentrations (10(-3) and 10(-2)M) of this nucleotide inhibited DNA synthesis in the lymphocytes of healthy subjects as well as cell patients. Impairment of 3H-thymidine incorporation observed in cll (in non-isolated cell populations) seems to be due to the presence of non-proliferating lymphocyte B population.     

Changes in human T lymphocytes after thymectomy and during senescence

Peripheral lymphocytes from individuals who had been thymectomized in adult life for myasthenia gravis (MG) or for other, nonimmunological reasons showed a moderate decrease in proliferative response capacity to several T-cell mitogens as compared to lymphocytes from normal individuals. The decrease of the response to mitogens and allogeneic lymphocytes was 20–30% within 5 years after thymectomy and about 50% more than 15 years after thymectomy. A comparable decrease in lymphocyte proliferative response capacity was found in healthy aged humans (68–97 years old). Analysis of T lymphocytes from both aged and thymectomized individuals with monoclonal (OKT) antibodies showed a similar pattern: the proportion of T lymphocytes binding OKT3 was reduced, and the OKT4/OKT8 ratio was increased. Hardly any T lymphocytes binding OKT6, OKT10, or OKT1 were found. A biochemical parameter for human T-cell differentiation, the lactate dehydrogenase (LDH) isoenzyme pattern, showed a significantly lower H/M ratio in the group of elderly people compared to young individuals. Furthermore, among patients thymectomized for MG, a significant correlation was observed between the LDH isoenzyme pattern of the T lymphocytes and the proliferative response to mitogens of these cells. In contrast, in healthy thymectomized individuals the LDH isoenzyme pattern appeared to be normal. These findings indicate that, after thymectomy or involution of the thymus, at least part of the peripheral blood T lymphocytes have properties different from those of the cells of young individuals. These cells might represent immature and/or not fully differentiated lymphocytes.     

Effect of Cyclosporin A on the Generation of Cytotoxic T Lymphocytes in Mouse Mixed Lymphocyte Culture

Cyclosporin A inhibited at equal concentrations both the proliferative response and the generation of cytotoxic T lymphocytes in one-way mouse spleen cell mixed lymphocyte culture. The 50% inhibitory concentration was in all experiments 10(-2) to 10(-1) microgram/ml. The inhibition was directly proportional to how early the drug was added to the culture: a complete inhibition of both responses was obtained if the drug was added on day 3, and a partial inhibition if added on day 4 of culture. Cytological analysis of the cultured cells demonstrated that resting lymphocytes were not damaged at 100-1000-fold concentrations of the drug giving complete inhibition of the blastogenic response. The results suggest that cyclosporin A is most effective if present throughout the induction phase of the immune response.     

Activation of Human T Lymphocytes by Soluble Protein A

The proliferative response of highly purified human peripheral blood or tonsil lymphocytes in the presence of soluble protein A (SpA) was investigated. SpA was shown to be a potent mitogen for T lymphocytes from peripheral blood or tonsils. Conversely, the non-T lymphocyte population from peripheral blood responded poorly to SpA, and SpA did not stimulate non-T lymphocytes from tonsils. The addition of mitomycin-treated T lymphocytes from peripheral blood enhanced the response of blood non-T lymphocytes to protein A. This effect was not found when non-T lymphocytes isolated from tonsils were cultured with mitomycin-treated T lymphocytes. The proliferative response of unseparated cells or purified lymphocyte populations was enhanced when adherent cells were added to the cultures. It is concluded that the soluble form of protein A activates mainly T lymphocytes and does not induce B lymphocyte proliferation.     

Effects of iron and culture filtrates on killing of Neisseria gonorrhoeae by normal human serum.

Neisseria gonorrhoeae GC9, both colony types T2 and T4, were killed by normal human serum, although populations of colony type T4 were more susceptible. Ferric ammonium citrate prevented the killing of populations of both T2 and T4 colony types. Other iron compounds tested showed no protective effect, nor did ammonium citrate or the divalent cations magnesium or calcium. A filtrate from cultures of an N. gonorrhoeae strain grown in a liquid defined medium showed a similar protective effect in the serum assay. The filtrate appeared to chelate iron, as measured by decreased ability of iron-free transferin to bind iron in the presence of the filtrate. However, the two effects did not appear to be related. Neither ferric ammonium citrate nor the culture filtrate sufficiently inactivated complement to account for protection.     

Mitogenic activity of extracellular cationic products produced by group A streptococci; analysis of the lymphocyte response.

The cationic fraction (isoelectric point greater than 8.5) of supernatant products of group A streptococcal cultures exerted a strong mitogenic effect on human peripheral lymphocytes at concentrations as low as 1 ng/well. Incorporation rates were highest at concentrations of 1-10 micrograms; rabbit peripheral lymphocytes also responded strongly, only a weak response was seen with mouse peripheral lymphocytes and rabbit thymocytes. Purified OKT4 positive (T helper) and OKT8 positive (T suppressor) lymphocyte subpopulations both responded, the former more strongly. Although accessory cells (monocytes) were not absolutely necessary, in their presence higher incorporation of 3H-thymidine was observed. Isolated B cells did not respond.     

Chronic lymphocytic leukaemia. Studies on mitogen-stimulated lymphocyte interferon as a new technique for assessing T lymphocyte effector function

The present study employs a new technique for the study of human T-cell effector function in patients with chronic lymphocytic leukaemia: mitogen-stimulated interferon. Cultures of macrophages, T cell-enriched lymphocytes, or macrophages and lymphocytes combined were prepared from the blood of fourteen normal donors and five patients with chronic lymphocytic leukaemia. The effects of the mitogens, phytohaemagglutinin and pokeweed, on interferon production and lymphocyte transformation were studied and the following observations made: (a) T-cell effector and proliferative functions were depressed as evidenced by the absence of interferon and proliferative response to PHA and PWM at 3 days in vitro; (b) Three out of five patients showed no interferon or proliferative response at 6 days, thus indicating a B lymphocyte abnormality as well; (c) macrophages from both normal and leukaemic subjects augmented mitogen-stimulated lymphocyte interferon production and lymphocyte transformation. However, the addition of normal allogeneic macrophages to cultures of lymphocytes prepared from the patients did not restore the proliferative and interferon responses to normal levels.     

The effect of non-transferrin-bound iron on murine T lymphocyte subsets: analysis by clonal techniques.

A number of different immunological properties have been attributed to iron (Fe3+ and Fe2+) and iron-binding proteins. However, in many previous studies, high concentrations of iron were used and cell-cell interactions were not excluded as a possible cause of the observed immunomodulatory effects. In this study, clonal techniques have been used to examine the effect of non-transferrin-bound iron (Fe3+) on the T lymphocyte subsets required for the generation of cytotoxic T lymphocytes (CTL). Concentrations of non-transferrin-bound Fe3+ of 10 microM or greater were shown to inhibit the generation of C57, BALB/c and CBA allo-specific CTL in bulk culture. Limit-dilution analysis revealed that: (i) Fe3+ reduced the cloning efficiency of CTL-precursors (CTL-P) by up to 96% without affecting the rate of clone growth; (ii) Fe3+ did not affect the cloning efficiency of allo-stimulated Ly-2-ve T cell precursors but reduced the rate of clone growth of these cells; (iii) Fe3+ enhanced, by more than 13-fold, the function of clones of Concanavalin A (Con A)-induced suppressor T lymphocytes (STL) which suppressed in vitro the development of CTL from their precursor cells. The data provide further evidence that low concentrations of non-transferrin-bound Fe3+, of the same order as those reported to be present in the serum of patients with iron-overload, have significant immunoregulatory properties.     

Differential responses to mitogen stimulation in lymphocytes from normal individuals and Lesch-Nyhan patients: influence of the bicarbonate buffer system.

Three patients affected with the Lesch-Nyhan syndrome were found to have normal levels of immunoglobulins, normal numbers of circulating B and T cells and normal IgG secretion in vitro in response to polyclonal activators. However, when cultures were performed in the absence of a bicarbonate buffer system, the proliferative response to several T cell stimulants (phytohaemagglutinin, concanavalin A and streptokinase-streptodornase) was impaired in Lesch-Nyhan cells as judged from the incorporation of labelled thymidine, uridine and leucine. This situation could be abolished by incubation in a 5% CO2 atmosphere and even reversed by supplementation of bicarbonate to the culture medium. Blocking the de novo purine synthesis by Methotrexate resulted in a more pronounced inhibition of the mitogenic response in Lesch-Nyhan lymphocytes than in normal cells. The differences in proliferative response between normal and Lesch-Nyhan lymphocytes with regard to culture conditions point to the critical role of the de novo pathway in lymphocyte stimulation.