Effects of mechanical stress on cytokine production in mandible-derived osteoblasts.

OBJECTIVE: Mechanical stress is known to be an important factor in the regulation of bone remodeling, and mandibular bone is continuously exposed to mechanical stressors such as occlusal force. Therefore, in this study, we investigated the effects of mechanical stress approaching occlusal force, to which mandible-derived osteoblasts (MDOB) are exposed, on cytokine expression and production using an original hydrostatic pressure apparatus. MATERIALS AND METHODS: The levels of cytokine in MDOB were examined by real-time RT-PCR, ELISA, and western blotting. In addition, mitogen-activated protein kinase inhibitor for ERK1/2, JNK, and p-38 pathways was used to identify the signal transduction pathway. RESULTS: Hydrostatic pressure increased the expression of IL-6 and TNF-α mRNA in a magnitude- and time-dependent manner and also enhanced IL-6 and TNF-α protein production. Furthermore, hydrostatic pressure changed the RANKL/OPG ratio in favor of RANKL for both mRNA and protein levels. Specific inhibitor of p-38 pathway but not that of the ERK1/2 and JNK pathways suppressed the up-regulation of RANKL production induced by hydrostatic pressure loading. CONCLUSION: These results suggest that MDOB play a role in cytokine production in response to mechanical stress and that occlusal force may support the maintenance of mandible bone homeostasis by activating bone remodeling through osteoclastogenesis.

Cytokine production in human periodontal ligament cells stimulated with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Although some functions and characterizations of human periodontal ligament (hPDL) cells have been reported, the role of hPDL cells in periodontal disease is poorly understood. We have previously reported that hPDL cells produce many kinds of inflammatory cytokines by stimulation with Prevotella intermedia. In this study, we examined the production of cytokines in hPDL cells stimulated with Porphyromonas gingivalis as compared with P. intermedia. MATERIAL AND METHODS: hPDL cells cultured in Dulbecco's modified Eagles's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics. After three to four passages, hPDL cells were stimulated with P. intermedia (ATCC25601) or P. gingivalis (ATCC33277) for 24 h. Total RNA was extracted by ISOGEN and the expression of cytokine mRNA was determined using reverse transcription-polymerase chain reaction. Cytokines in the culture supernatants were assessed by enzyme-linked immunosorbent assay. RESULTS: The expression of interleukin-1beta, interleukin-6, interleukin-8, tumor necrosis factor-alpha (TNF-alpha), receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) mRNA was detected in hPDL cells after stimulation with P. gingivalis as well as P. intermedia. There were no significant differences in the kind of cytokines expressed in hPDL cells between P. gingivalis and P. intermedia. However, P. gingivalis induced a significantly higher production of cytokines in hPDL cells than P. intermedia (p < 0.05). CONCLUSION: This study demonstrated that hPDL cells produce many kinds of cytokines as a result of bacterial stimulation, including stimulation with P. gingivalis and P. intermedia. These results suggest that hPDL cells may play a role in cytokine production in periodontal disease.

Mechanical stress induces expression of cytokines in human periodontal ligament cells.

OBJECTIVE: Periodontal tissue has a unique structure in that the human periodontal ligament (hPDL) lies between the hard tissues of cementum and alveolar bone. Although the role of cytokines in hPDL function is not clearly understood, we investigated the effect of mechanical stress as hydrostatic pressure (HP) on cytokine expression in hPDL cells. MATERIALS AND METHODS: The hPDL cells were obtained from a healthy maxillary third molar. After the third to fourth passage, the cells were exposed to HP ranging from 1 to 6 MPa as previously described. Total RNA was extracted and the expression of cytokine mRNA was determined by RT-PCR. RESULTS: The exposure to 6 MPa of HP caused no morphological changes of hPDL cells, and did not affect the cellular viability. No expression of IL-1beta, IL-6, IL-8, TNF-alpha, receptor activator of NF-lambdaB (RANK), receptor activator of NF-lambdaB ligand (RANKL), or osteoprotegerin mRNA was observed in the control cells under atmospheric pressure, whereas, in hPDL cells treated with HP, a pressure-dependent enhancement of IL-6, IL-8, RANKL, and OPG mRNA expression was observed between 10 and 60 min after the exposure to HP. CONCLUSION: These results suggest that hPDL cells may play a role in the production of cytokines in response to mechanical stress in vivo.

A role for ferritin in hematopoiesis and the immune system.

Elevated serum ferritin levels have been reported in a number of pathological states. These observations indicate that cells of the immune system can participate in the prevention of potential tissue toxicity from iron accumulation, and iron and iron-binding protein have important effects on immune systems. Ferritin is generally regarded as an intracellular iron storage protein. However, small amounts of ferritin circulate in the serum of normal individuals, and the physiological role of serum ferritin remains obscure. Although the function of ferritin is inevitably linked to iron metabolism, a role for ferritin in hematopoiesis and the immune system has drawn attention for years. Ferritin has an inhibitory effect on the in vitro growth of human hematopoietic progenitor cells and on the proliferation of T lymphocytes in vitro. Recently we report that ferritin may directly suppress the differentiation of human B lymphocytes maturing into antibody producing cells in vitro. In the present review, we summarise this field of research.

Distinct effect of G-CSF on the growth and differentiation of myeloid progenitor cells from chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) is a disorder characterized by severe neutropenia and a maturational arrest of the neutrophil precursors in the bone marrow. We examined the effect of recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the growth and maturation of the myeloid progenitor cells from a patient with CIN. The patient's marrow cells showed poor colony forming activity, but a normal differentiating capacity to the stimulation with rhG-CSF, although they displayed a normal colony forming capacity in the presence of GM-CSF. Our observation indicates the distinct effect of rhG-CSF on the growth and maturation of the myeloid progenitors from a CIN patient.

Immunomodulatory effects of three macrolides, midecamycin acetate, josamycin, and clarithromycin, on human T-lymphocyte function in vitro.

The effect of three macrolide antibiotics, midecamycin acetate, josamycin, and clarithromycin, on human T-cell function was investigated in vitro. Midecamycin acetate and josamycin suppressed the proliferative response of peripheral blood mononuclear cells stimulated by polyclonal T-cell mitogens at concentrations between 1.6 and 8 micrograms/ml. At higher concentrations (40 to 200 micrograms/ml), all these drugs showed a marked inhibitory effect. At concentrations of 1.6 to 40 micrograms/ml, these drugs suppressed interleukin-2 (IL-2) production induced by mitogen-stimulated T cells, but not the expression of IL-2 receptor (CD25), in a dose-dependent manner. Therefore, the suppressive action on T-lymphocyte proliferation seems to be based on the ability of these drugs to inhibit IL-2 production by T cells. The drug also inhibited mixed lymphocyte reaction at the same concentrations. Combined treatment with these macrolides and the known immunosuppressants such as FK506 and cyclosporin A resulted in an increased inhibition of T-cell proliferation. The immunomodulatory properties of the antibiotics may have clinical relevance for modulation of the immune response in transplant patients and in patients with inflammatory diseases.

Immunosuppressive activity of bromocriptine on human T lymphocyte function in vitro.

Bromocriptine (BRC), a dopamine type 2 agonist, prevents secretion of pituitary prolactin (PRL). BRC has been shown to impair lymphocyte responsiveness toward antigenic stimulation by decreasing serum PRL levels. Hypoprolactinaemia induced by BRC produces a similar immunosuppressive effect, as observed in hypophysectomized rats, which is restored by the administration of PRL. Therefore, the immunosuppression induced by BRC has been interpreted as the result of hypoprolactinaemia. However, the direct mechanism of BRC in immune response has never been evoked. We recently reported that BRC has an immunosuppressive activity on human B lymphocyte function in vitro. In the present study we demonstrate that BRC suppresses T cell proliferation by means of blocking IL-2 production by T cells as well as mixed lymphocyte reaction (MLR) in a dose-dependent manner. We could not detect the immunoreactive PRL activity in the conditioned medium from polyclonal T cell mitogen-stimulated T cell cultures. Then, the immunosuppressive activity of BRC on human T cell function appeared to be independent of its hypoprolactinaemic effect. Treatment with low-dose cyclosporin A (CsA) or FK506 in combination with BRC has proved more effective than either drug alone in suppression of T cell proliferation and CD25 antigen expression. Thus, the therapeutic application of BRC in combination with immunosuppressants may enhance the immunosuppressive effect, while at the same time decreasing the toxicity.

H- and L-rich ferritins suppress antibody production, but not proliferation, of human B lymphocytes in vitro.

The effect of human spleen(L-rich) and heart(H-rich) ferritins on the proliferation and differentiation of human B lymphocytes was studied in comparison with that of holo- and apo-transferrins. Ferritins rich in H and L chain, as well as the transferrins, did not inhibit the proliferative response of resting and activated B cells stimulated with polyclonal B-cell mitogen, Staphylococcus aureus Cowan strain I. In contrast, the ferritins, but not the transferrins, clearly suppressed the antibody production by B blasts in T-cell-independent as well as T-cell-dependent system. Kinetic study showed that inhibitory action of ferritins on immunoglobulin (Ig) production was caused at an early stage of B-cell differentiation. The cytoplasmic Ig-containing cells decreased in proportion to the reduction of Ig secretion. The evidence that ferritin inhibited Ig synthesis of Epstein-Barr virus-transformed human B-lymphoblastoid cell line also supported the idea that the effect of ferritin was directed toward the antibody-producing B lymphocytes. The molecular analysis showed that the inhibitory effect of ferritin was regulated at the transcriptional level of the Ig generation signal. Our results suggest that H- and L-rich ferritins exert their inhibitory action on the differentiation of B cells maturing into Ig-producing cells.

Immunosuppressive activity of fosfomycin on human T-lymphocyte function in vitro.

Recent investigations have shown that some antibiotics also work as immunomodulators. We have recently reported that fosfomycin (FOM) has an immunomodulatory effect on human B-cell activation. FOM is a unique antibiotic which is chemically unrelated to any other known antibacterial agent. In the present study, we examined the effect of FOM on human T-cell function. FOM inhibited the proliferation of human lymphocytes induced by polyclonal T-cell mitogens in a dose-dependent manner. FOM also strongly suppressed mixed lymphocyte reaction and interleukin-2 (IL-2) production by T cells. Moreover, FOM inhibited the expressions of IL-2 receptor (CD25) and transferrin receptor (CD71) on the activated T-cell surfaces. These data suggest that FOM may block the T-cell division during the transition from G1 to S phase of the cell cycle. Combined treatment with FOM and low-dose cyclosporin A or FK506 caused additive or synergistic suppression of T-cell proliferation, but not on IL-2 receptor expression. It seems that the mode of action of FOM on T-cell function involves a specific suppression of IL-2 production.

[Biosynthesis and secretory regulation of pituitary prolactin].

We summarized recent advancements in biosynthesis and secretory regulation of pituitary prolactin at the cellular and molecular levels from some literatures. We singled out dopamine, estrogen and thyrotropin releasing hormone (TRH) from many factors controlling the biosynthesis and the regulation, because they are important, physiologically and pathologically. Dopamine exerts some effects on the membranes of the lactotrope inhibiting activities of cAMP, phosphatidylinositols and calcium ion channels. It inhibits prolactin gene transcription by suppressing action of the promoter region. Estrogen and TRH increase prolactin biosynthesis and the secretory regulation. Estrogen coupled with the receptors in the cytoplasma goes directly to the DNA region -1713-->-1495 being upstream to the starting site of prolactin transcription and differing from the region on which dopamine acts. TRH accelerates prolactin production and release in such steps as transcription, stabilization of mRNA, accumulation of mRNA in the cytoplasma, protein synthesis and exocytosis.

G-CSF enhances the immunoglobulin generation rather than the proliferation of human B lymphocytes.

The effect of granulocyte-colony stimulating factor (G-CSF) on human B-cell function was studied in in vitro cultures. G-CSF alone had no effect on the proliferative response of resting B cells, but it slightly enhanced the proliferative response of these cells in the presence of polyclonal B-cell mitogen, Staphylococcus aureus Cowan strain I (SAC) at concentrations of 0.2 to 25 micrograms/ml (1.5-fold increase in the DNA synthesis). In contrast, immunoglobulin (Ig) secretion of activated B cells was increased approximately three-fold to four-fold by adding G-CSF to the cultures. The neutralization of G-CSF bioactivity with anti-G-CSF antibody abrogated this effect. Though cytoplasmic Ig-positive cells or plasma cell marker-positive cells did not change, the expression of IgM mRNA in antibody-producing B cells increased in the presence of G-CSF in the cultures. Interestingly, human B lymphocytes are shown to express the binding to biotin-conjugated G-CSF preparation, but not to biotin-conjugated GM-CSF preparation when examined by flow cytometry. These data suggest that G-CSF may influence B-cell function in special circumstances.

Impaired G-CSF production at post-transcriptional level in a patient with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) is a disorder characterized by severe neutropenia and a maturational arrest of the neutrophil precursors in the bone marrow. The pathogenesis of this disorder has been obscure. We examined the production of endogenous G-CSF and the expression of G-CSFmRNA in a patient with adult type CIN. The G-CSF production by the patient's mononuclear cells was deficient in spite of the adequate accumulation of G-CSFmRNA. Our data suggests that the defect in the endogenous G-CSF production at the post-transcriptional level is likely to be an aetiological factor in CIN.

Induction of CD5 antigen on human CD5- B cells by stimulation with Staphylococcus aureus Cowan strain I.

We have examined whether the CD5 phenotype could be induced on human B cell surfaces by the polyclonal B cell stimulator, Staphylococcus aureus Cowan strain I (SAC). Fresh tonsillar B cells were prepared by Percoll density gradient from E- cells. The proportion of CD5+ B cells in the 50/60% and 60/70% interface high-density fractions varied between 1.2 and 10.2% depending on the tonsil preparations when they were placed on the in vitro culture 12-60 h prior to flow cytometric analysis. The expression of CD5 antigen obviously increased in the presence of SAC (1:10(5) v/v). The percentage of CD5+ B cells varied from tonsil to tonsil, from 25.1 to 65.9% in a series of experiments. The CD5+ B cells were found both among CD23+CD25+CD71+ and CD23-CD25-CD71- B cells. The level of CD5 expression was related to the cell size enlargement. The addition of anti-CD5 antibody in the culture blocked the CD5 induction by SAC without interfering with the expression of other activation markers. A time-course study showed that CD5 antigen appeared to be induced on the cell surface during the G0 to G1 phase transition in the cell cycle. When CD5+ and CD5- B cells were separated by magnetic isolation, the CD5- B cells showed DNA synthesis to the stimulation by SAC and expressed CD5 antigen on their cell surface. These results suggest that human CD5- B cells can express the CD5 phenotype by stimulation with the polyclonal B cell stimulator, SAC.

Immunosuppressive property of bromocriptine on human B lymphocyte function in vitro.

Bromocriptine (BRC), a dopamine ergot alkaloid, inhibits the release of pituitary prolactin (PRL). Hypoprolactinaemia induced in rat by treatment with BRC produces a similar immunosuppressive effect as observed in hypophysectomized rats. The effect of immunosuppression by the administration of BRC has been interpreted as the result of hypoprolactinaemia produced by BRC. However, the direct effect of BRC on lymphocyte function has never been evaluated. The purpose of this study was to investigate the in vitro effect of BRC on human B cell functions. Highly purified B cells from tonsil samples were isolated by Percoll density gradient from non-rosetted cells, and were used as target cells. BRC significantly suppressed the proliferative response of resting and activated B cells in vitro. It suppressed immunoglobulin generation of activated B cells. The inhibition of BRC was manifested in the early stage of the proliferation and differentiation of B cells. The conditioned medium from the polyclonal B cell mitogen-stimulated B cell cultures did not contain PRL as detected by immunoradiometric assay. Treatment with low-dose cyclosporin A or FK506 in conjunction with BRC has proved more effective than either drug alone in suppression of B cell proliferation. Thus, the combined therapy of BRC and immunosuppressants may be effective with decreased toxicity for clinical use.

Recombinant human IL-5 augments immunoglobulin generation by human B lymphocytes in the presence of IL-2.

The biological activity of interleukin-5 (IL-5) on human B lymphocytes was examined by using a newly purified recombinant human IL-5 preparation. Different densities of B cells from human tonsil samples were isolated by Percoll density gradient from nonrosetted cells. The IL-5 preparation did not act as a B cell growth factor on the large (in vivo-activated) or small (resting) B cells after activation with polyclonal B cell activators. The IL-5 augmented the immunoglobulin (Ig) generation of Staphylococcus aureus Cowan strain I (SAC)-activated B blasts in the presence of interleukin-2 (IL-2). The effect of IL-5 on Ig generation was greater in pokeweed mitogen (PWM)-driven B blasts than that in SAC-induced B blasts when assessed by enzyme-linked immunosorbent assay. The amounts of mu RNA isolated from IL-5/PWM-treated cells were also larger than those from IL-5/IL-2-treated B blasts. These results suggest that the human IL-5 can increase Ig generation of B cells in the presence of IL-2 by working at the level of mRNA coding for Ig.

Immunomodulatory effect of fosfomycin on human B-lymphocyte function.

Fosfomycin (FOM) is an unique antibiotic which is chemically unrelated to any other known antimicrobial agent. Recent investigations have demonstrated that FOM inhibits histamine release from basophils. In this study, we examined the effect of FOM on human B-cell functions. FOM inhibited the proliferative response of resting B cells induced by Staphylococcus aureus Cowan 1 in a dose-dependent manner. FOM interfered with the transition from the G0 to the G1 phase of the cell cycle, leading to cell arrest. The proliferative response of in vivo-activated B cells and lymphokine-induced B-cell proliferation were also affected by FOM. In addition, FOM suppressed immunoglobulin secretion by antibody-producing B cells. Interestingly, FOM did not affect the expression of activation antigens such as the CD25 (interleukin-2 receptor) and CD71 (transferrin receptor) antigens. Moreover, FOM sustained the increased Ia expression on B-cell membranes induced by S. aureus Cowan 1 stimulation, which suggests that FOM may not block the role of B cells in antigen presentation in T-cell-B-cell interaction.

Bromocriptine effects on plasma luteinizing hormone and its responses to gonadotropin-releasing hormone in normal men.

Secretory changes in plasma pituitary luteinizing hormone (LH) after administration of a dopaminergic drug were studied in five normal men. Each subject received orally 5 mg of bromocriptine (Brc) daily for 8 weeks. Each received gonadotropin-releasing hormone (GnRH) stimulation tests at the beginning (control), and at 2, 4, 6, and 8 weeks after initiation of Brc treatment. We referred the basal plasma LH and a ratio of maximally GnRH-responded plasma LH to its basal level (R-Max) as indicators of secretory alterations of the LH. Mean basal levels of plasma LH in the five subjects at the beginning and those of R-Max were 3.4 +/- 2.3 (SD) mIU/mL and 8.5 +/- 2.9 units, respectively. Statistically, both the mean values of plasma LH and R-Max during the control period did not differ significantly from those obtained after Brc treatment, although mean basal levels of plasma prolactin during the control period (15.6 +/- 4.6 ng/mL) decreased significantly (p < 0.05) after initiation of treatment. A low dose of Brc administered to normal men for 8 weeks does not significantly influence pituitary secretion of LH.

The distinct effects of FK506 on the activation, proliferation, and differentiation of human B lymphocytes.

We examined the effect of FK506 on the activation, proliferation and differentiation of human B lymphocytes in vitro. FK506 inhibited the proliferative response of resting B cells induced by Staphylococcus aureus Cowan strain I (SAC) and phorbol myristate acetate (PMA) in a dose-dependent manner. Inhibition of cell proliferation by FK506 was caused by a selective block of G0 to G1 phase transition leading to cell arrest. In addition, the proliferative response of in vivo-activated B cells and lymphokine-driven B cell proliferation were also found to be sensitive to FK506. Interestingly, FK506 did not affect the expression of activation antigens such as CD23, IL-2 receptor (CD25), and transferrin receptor (CD71). Finally, FK506 had little effect on B cell antibody generation in a T cell-independent system. Conversely, FK506 suppressed neither proliferation nor immunoglobulin secretion in a human B lymphoblastoid cell line. These results indicate that FK506 has discrete effects on the different stages of the B cell maturation.

The suppressive effect of deoxyspergualin on the differentiation of human B lymphocytes maturing into immunoglobulin-producing cells.

Deoxyspergualin, an analog of spergualin, has been known as a novel immunosuppressive agent with strong immunosuppressive activity in in vivo experimental systems. In the present study, we examined the effect of deoxyspergualin (DSG) and methyldeoxyspergualin (MeDSG) on the proliferation and differentiation of human B lymphocytes in vitro. Highly purified B cells from human tonsil samples were isolated by Percoll density gradient from nonrosetted cells and were used as target cells. Both agents had little effect on the proliferative response of resting or activated B lymphocytes. However, they suppressed the immunoglobulin synthesis of B lymphocytes not only in a T cell-dependent, but also in a T cell-independent system. The inhibition of antibody synthesis was manifested in the early stage of B cell differentiation. Both drugs also suppressed Ig secretion, but not proliferation, of an EBV-transformed human B lymphoblastoid cell line. These results indicate that DSG and MeDSG have a selective immunosuppressive effect on the differentiation pathway of B lymphocytes.