Low-Avidity Autoantibodies against Bactericidal/Permeability-Increasing Protein Occur in Gram-Negative and Gram-Positive Bacteremia.

Antibody autoreactivity against bactericidal/permeability-increasing protein (BPI) is strongly associated with Pseudomonas aeruginosa infection in cystic fibrosis (CF), non-CF bronchiectasis (BE), and chronic obstructive pulmonary disease (COPD). We examined the pathogen-specific nature of this autoreactivity by examining antibodies to BPI in bacteremia patients. Antibodies to BPI and bacterial antigens were measured in sera by ELISA from five patient cohorts (n = 214). Antibody avidity was investigated. Bacteremic patient sera (n = 32) exhibited IgG antibody autoreactivity against BPI in 64.7% and 46.7% of patients with positive blood cultures for P. aeruginosa and Escherichia coli, respectively. Autoantibody titers correlated with IgG responses to bacterial extracts and lipopolysaccharide (LPS). A prospective cohort of bacteremic patient sera exhibited anti-BPI IgG responses in 23/154 (14.9%) patients with autoreactivity present at the time of positive blood cultures in patients with Gram-negative and Gram-positive bacteria, including 8/60 (13.3%) patients with Staphylococcus aureus Chronic tissue infection with S. aureus was associated with BPI antibody autoreactivity in 2/15 patients (13.3%). Previously, we demonstrated that BPI autoreactivity in CF patient sera exhibits high avidity. Here, a similar pattern was seen in BE patient sera. In contrast, sera from patients with bacteremia exhibited low avidity. These data indicate that low-avidity IgG responses to BPI can arise acutely in response to bacteremia and that this association is not limited to P. aeruginosa This is to be contrasted with chronic respiratory infection with P. aeruginosa, suggesting that either the chronicity or the site of infection selects for the generation of high-avidity responses, with biologic consequences for airway immunity.

Inhibition of joint damage and improved clinical outcomes with rituximab plus methotrexate in early active rheumatoid arthritis: the IMAGE trial.

OBJECTIVES: Rituximab is an effective treatment in patients with established rheumatoid arthritis (RA). The objective of the IMAGE study was to determine the efficacy of rituximab in the prevention of joint damage and its safety in combination with methotrexate (MTX) in patients initiating treatment with MTX. METHODS: In this double-blind randomised controlled phase III study, 755 MTX-naïve patients with active RA were randomly assigned to MTX alone, rituximab 2×500 mg + MTX or rituximab 2×1000 mg + MTX. The primary end point at week 52 was the change in joint damage measured using a Genant-modified Sharp score. RESULTS: 249, 249 and 250 patients were randomly assigned to MTX alone, rituximab 2×500 mg + MTX or rituximab 2×1000 mg + MTX, respectively. At week 52, treatment with rituximab 2×1000 mg + MTX compared with MTX alone was associated with a reduction in progression of joint damage (mean change in total modified Sharp score 0.359 vs 1.079; p=0.0004) and an improvement in clinical outcomes (ACR50 65% vs 42%; p<0.0001); rituximab 2×500 mg + MTX improved clinical outcomes (ACR50 59% vs 42%; p<0.0001) compared with MTX alone but did not significantly reduce the progression of joint damage. Safety outcomes were similar between treatment groups. CONCLUSIONS: Treatment with rituximab 2×1000 mg in combination with MTX is an effective therapy for the treatment of patients with MTX-naïve RA. ClinicalTrials.gov identifier NCT00299104.

Efficacy and safety of different doses and retreatment of rituximab: a randomised, placebo-controlled trial in patients who are biological naive with active rheumatoid arthritis and an inadequate response to methotrexate (Study Evaluating Rituximab's Efficacy in MTX iNadequate rEsponders (SERENE)).

OBJECTIVES: This phase III study evaluated the efficacy and safety of rituximab plus methotrexate (MTX) in patients with active rheumatoid arthritis (RA) who had an inadequate response to MTX and who were naïve to prior biological treatment. METHODS: Patients with active disease on stable MTX (10-25 mg/week) were randomised to rituximab 2 x 500 mg (n=168), rituximab 2 x 1000 mg (n=172), or placebo (n=172). From week 24, patients not in remission (Disease Activity Score (28 joints) > or =2.6) received a second course of rituximab; patients initially assigned to placebo switched to rituximab 2 x 500 mg. The primary end point was American College of Rheumatology 20 (ACR20) response at week 24. All patients were followed until week 48. RESULTS: At week 24, both doses of rituximab showed statistically superior efficacy (p<0.0001) to placebo (ACR20: 54%, 51% and 23%; rituximab (2 x 500 mg) + MTX, rituximab (2 x 1000 mg) + MTX and placebo + MTX, respectively). Secondary end points were also significantly improved for both rituximab groups compared with placebo. Further improvements in both rituximab arms were observed from week 24 to week 48. Rituximab + MTX was well tolerated, demonstrating comparable safety to placebo + MTX through to week 24, and between rituximab doses through to week 48. CONCLUSIONS: Rituximab (at 2 x 500 mg and 2 x 1000 mg) plus MTX significantly improved clinical outcomes at week 24, which were further improved by week 48. No significant differences in either clinical or safety outcomes were apparent between the rituximab doses.

Dissociation of systemic and mucosal autoimmunity in cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa accounts for ~80% of cystic fibrosis (CF) airway infection. It shows a remarkable correlation with presence of autoantibody to bactericidal/permeability-increasing protein (BPI), which is not understood. In this study, we sought to better understand the characteristics of systemic and mucosal autoimmunity and their relation to humoral immunity to P. aeruginosa. METHODS: Antibody titers and isotypes to BPI and P. aeruginosa were characterized in sera and bronchoalveolar lavage (BAL) of adult and pediatric CF patients (n = 131), by ELISA and/or immunoblot. RESULTS: Serum BPI autoantibodies were common (~43%) in adult while rare (≪5%) in pediatric (≤18 yrs) CF patients. Serum BPI IgG autoantibodies were of high avidity and strongly correlated with anti-P. aeruginosa IgG responses. A parallel relationship was observed with IgA, but not IgG, responses in adult and pediatric CF patient in the BAL. Thus, BAL IgA anti-BPI antibodies were independent of age and correlated with the presence of BPI cleavage in BAL. CONCLUSIONS: IgG and IgA autoreactivity to BPI in CF patients was demonstrated in serum and BAL, respectively, and correlated with the isotype of the antibody response to P. aeruginosa. The co-occurrence of anti-BPI and anti-P. aeruginosa IgA in the BAL, but not serum, of pediatric CF patients suggests that BPI tolerance is broken in the P. aeruginosa-infected airway and that serologic IgG autoantibodies are later induced, potentially through a separate pathway. The relationship between P. aeruginosa, BPI cleavage, and IgA autoantibodies in the BAL suggests a role for cryptic epitope generation in the breaking of tolerance.

Regulation of lymphokine production and human T lymphocyte activation by 1,25-dihydroxyvitamin D3. Specific inhibition at the level of messenger RNA.

The steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (calcitriol), has been shown to inhibit T cell proliferation, primarily through inhibition of interleukin 2 (IL-2) production. In these experiments, we show that calcitriol also markedly inhibited production of the lymphokine, gamma interferon (IFN-gamma), by activated human T lymphocytes. Regulation of both IL-2 and IFN-gamma production as well as transferrin receptor (TfR) expression by calcitriol was apparent at the messenger RNA (mRNA) level as determined by Northern blotting. The decrease in IL-2 and IFN-gamma mRNA that occurred with calcitriol treatment was coordinate and not apparent up to 12 h after phytohemagglutinin stimulation, whereas decreased accumulation of TfR mRNA was not present before 24-36 h. Furthermore, the effects of calcitriol on IL-2, IFN-gamma, and TfR mRNA accumulation were specific; actin mRNA accumulation was comparable between control and treated cells. These data indicate that calcitriol regulated proteins associated with T cell activation at the transcriptional level and that these effects were mediated in a specific, coordinate fashion.

Inhibition of T lymphocyte mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol).

Recent studies have suggested that vitamin D may have other important biologic activities in addition to its well-characterized role in the maintenance of calcium homeostasis. Discovery of cytosolic receptors for vitamin D in human peripheral blood monocytes and lectin-stimulated lymphocytes prompted us to study the effects of 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, upon phytohemagglutinin (PHA)-induced lymphocyte blast transformation. We have found that calcitriol is a potent inhibitor of PHA-induced lymphocyte proliferation, achieving 70% inhibition of tritiated thymidine incorporation after 72 h in culture. Furthermore, calcitriol suppressed interleukin-2 (IL-2) production by PHA-stimulated peripheral blood mononuclear cells in a concentration-dependent fashion. Lastly, the suppressive effect of calcitriol on cellular proliferation was partially reversed by the addition of saturating amounts of purified IL-2. We conclude that calcitriol is a potent inhibitor of PHA-induced lymphocyte blast transformation and that this effect is mediated, in part, through suppression of IL-2 production. Thus, calcitriol appears to possess immunoregulatory properties that have been unappreciated heretofore.

Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique.

Post-transcriptional regulation is an important mechanism in cellular response to stimuli, allowing for the rapid and discrete expression of relevant proteins. Genes regulated by this mechanism have specific cis -acting elements, frequently in their 3' untranslated regions (UTRs), that have been shown to serve as recognition sites for trans -acting RNA-binding proteins. Unfortunately, the identification of specific mRNA ligands for different RNA binding proteins in vivo has been limited by a lack of adequate methodology. We have developed a novel technique that addresses this shortcoming, using immunoprecipitation of RNA binding proteins from polysomes followed by RT-PCR and library screening to identify the in vivo mRNA ligands of RNA binding proteins. Utilizing this approach, we have identified 32 known and 16 novel mRNAs specifically bound by the heterogeneous nuclear ribonucleoprotein (hnRNP) A2. Of the clones identified, 74% contained AU-rich elements and/or poly-uridine tracts in their 3' UTRs, cis -acting elements that have been established as impacting mRNA stability. The high percentage of clones containing these uridine-rich sequences compares favorably with the high affinity binding of poly-uridine RNA by hnRNP A2 in vitro. These data thus support the representative nature of the technique.

1,25-Dihydroxyvitamin D3 induces a myelomonocytic phenotype with enhanced effector cell function in the HL-60 promyelocytic leukemia cell line.

Human promyelocytic leukemia (HL-60) cells were induced by 1,25-dihydroxyvitamin D3 (calcitriol) to differentiate and examined using a panel of monoclonal antibodies (MoAbs) and functional assays. Although morphologically and histochemically these cells appeared to be of the monocyte-macrophage phenotype, there was a decline in Fc receptors for IgGl and no induction of class II HLA antigens. There was, however, dramatic induction of the antigen detected by the myeloid-specific MoAb AML-2-23. These data suggest that the phenotypic changes induced by calcitriol in HL-60 cells are consistent with myelomonocytic differentiation in that the resultant cells possess characteristics of both monocytes (morphology, non-specific esterase staining, high levels of AML-2-23 reactivity) and granulocytes (PMN 29 binding, decreased Fc receptors for IgGl, absence of class II HLA antigens). Perhaps more important, the ability of calcitriol-treated cells to perform antibody-dependent cellular cytotoxicity and phagocytosis was markedly augmented. Lysis of antibody-coated erythrocytes by HL-60 cells increased from 5% in controls to 30-35% with calcitriol treatment for 4 days. This enhanced effector cell function was seen despite a decline in Fc receptors measured by cytofluorography. These data suggest that calcitriol may be involved in both differential and functional activation of myeloid cells.

IgA-mediated effector function of HL-60 cells following treatment with calcitriol.

When cells of the HL-60 promyelocytic leukemia line are cultured with 1,25-dihydroxyvitamin D3 (calcitriol) they acquire a more highly differentiated, myelomonocytic phenotype. It was observed that the ability to ingest IgA-coated erythrocytes and to bind soluble dimeric IgA accompanied this maturation. Phagocytosis of IgA-coated erythrocytes was greater than 50% inhibited by 0.8 mg/ml free IgA, and not by IgG or IgM. Similarly, binding of dimeric IgA was not blocked by a 100-fold excess of IgG, IgM or IgE. Both IgA-mediated phagocytosis and IgA binding became apparent after two days of culture with calcitriol and increased with time in culture. The induction of functional IgA receptors was evident with 10(-11) M calcitriol and maximal levels of IgA binding and of numbers of cells capable of IgA mediated phagocytosis were induced by 10(-8)-10(-9) M calcitriol. 25-Hydroxyvitamin D3, which binds 100-1000-fold less avidly to the cytoplasmic D3 receptor than calcitriol, did not induce functional IgA receptors unless concns of 10(-7) M were used. Other compounds which induce differentiation of HL-60 cells, including retinoic acid and DMSO, produced similar results to calcitriol, whereas cells treated with gamma interferon expressed lower levels of IgA binding and did not ingest IgA-coated targets, suggesting that a critical density of IgA receptors must be reached to enable phagocytosis and/or that other cell activational events are required for IgA receptors to mediate killing. This model may provide useful insight into the function and regulation of IgA receptors on cells of the myeloid series.

The von Hippel-Lindau protein interacts with heteronuclear ribonucleoprotein a2 and regulates its expression.

The product of the von Hippel-Lindau (VHL) tumor suppressor gene, pVHL, functions as a ubiquitin-protein isopeptide ligase in regulating HIF-1 protein turnover, thus accounting for the increased transcription of hypoxia-inducible genes that accompanies VHL mutations. The increased vascular endothelial growth factor mRNA stability in cells lacking pVHL has been hypothesized to be due to a similar regulation of an RNA-binding protein. We report the expression of the GLUT-1 3'-untranslated region RNA-binding protein, heteronuclear ribonucleoprotein (hnRNP) A2, is specifically increased in pVHL-deficient cell lines. Enhanced hnRNP A2 expression was apparent in all cell fractions, including polysomes, where a similar modest effect on hnRNP L (a GLUT-1 and VEGF 3'-untranslated region-binding protein), was seen. Steady state levels of hnRNP A2 mRNA were unaffected. Regulation of hnRNP A2 levels correlated with the ability of pVHL to bind elongin C. Proteasome inhibition of cells expressing wild type pVHL selectively increased cytoplasmic hnRNP A2 levels to that seen in pVHL-deficient cells. Finally, an in vivo interaction between pVHL and hnRNP A2 was demonstrated in both the nucleus and the cytoplasm. Collectively, these data indicate that hnRNP A2 expression is regulated by pVHL in a manner that is dependent on elongin C interactions as well as functioning proteasomes.

1 alpha,25-dihydroxyvitamin D3 inhibits productive infection of human monocytes by HIV-1.

Pretreatment of freshly isolated human peripheral blood monocytes with the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)D), markedly reduced (by 95%) productive infection of human monocytes by HIV-1. Equivalent concentrations (10nM) of 25-hydroxyvitamin D3 (25(OH)D), the biologic precursor of 1,25(OH)D, were ineffective at reducing either CD4 expression or HIV-1 production. Pretreatment was required for modulation of HIV-1 infection by 1,25(OH)D. Interestingly, 1,25(OH)D-mediated decreases in p24 antigen production were observed prior to any observed reduction in CD4 expression, suggesting that 1,25(OH)D treatment may modulate HIV-1 infection of monocytes through additional factors besides decreased HIV-1 binding. These data raise the possibility that 1,25(OH)D compounds may be important in host resistance to HIV-1.

Decreased accessory cell function and costimulatory activity by 1,25-dihydroxyvitamin D3-treated monocytes.

To characterize the mechanism(s) by which 1,25-dihydroxyvitamin D3 (calcitriol) modulates the costimulatory capacity of monocytes, we examined the effect of calcitriol pretreatment of monocytes on their capacity to promote T cell proliferation (accessory cell function). Correlation of calcitriol-dependent changes in monocyte accessory cell function and alterations in phenotype and cytokine production, and the dependence of these changes on cell viability, were studied. Calcitriol pretreatment induced a defect in accessory cell function that was evident with fixed monocytes, suggesting a cell-surface-associated mechanism. Altered accessory cell function did not correlate with changes in HLA-DR antigen expression and was unaffected by concurrent treatment with interferon-gamma. Calcitriol treatment did not alter either the expression of adhesion molecules or monocytic production of interleukin-1 beta (IL-1 beta) or IL-6. Exogenous IL-1 or IL-6 did not overcome the impaired costimulatory activity of calcitriol-treated monocytes. Thus, calcitriol treatment reduces the capacity of monocytes to promote lectin-induced T cell activation at the level of the plasma membrane, perhaps through altered expression of an uncharacterized molecule important in monocyte-T cell interactions. At chronically inflamed sites, elaboration of calcitriol by activated macrophages may regulate the ability of monocytes to induce both antigen-dependent and antigen-independent T cell proliferation.

Glyceraldehyde-3-phosphate dehydrogenase selectively binds AU-rich RNA in the NAD(+)-binding region (Rossmann fold).

A 36-kDa protein that binds AU-rich RNA was purified from human spleen and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH has been previously demonstrated to bind tRNA with high affinity. Competition studies suggested that cytoplasmic GAPDH binds the AU-rich elements (AREs) of lymphokine mRNA 3'-untranslated regions with higher affinity than tRNA. The AUUUA-specific RNA binding activity of GAPDH was inhibited by NAD+, NADH, and ATP in a concentration-dependent manner, suggesting that RNA binding of GAPDH might involve the NAD(+)-binding region, or dinucleotide-binding (Rossmann) fold. This hypothesis was supported by experiments that localized RNA binding to the predicted N-terminal 6.8-kDa peptide, known to be involved in the formation of the NAD(+)-binding domain. The direct demonstration of ARE-specific binding protein activity localized to the NAD(+)-binding region of GAPDH supports the general concept that enzymes containing this domain may exhibit specific RNA binding activity and play additional roles in nucleic acid metabolism. Finally, cytoplasmic GAPDH was found in the polysomal fraction of T lymphocytes. Thus, the RNA binding specificity of GAPDH as well as its localization within the cell merit its strong consideration as a protein important in the regulation of ARE-dependent mRNA turnover and translation in addition to its well described role in glycolysis.

Characterization of RNA binding proteins associated with CD40 ligand (CD154) mRNA turnover in human T lymphocytes.

CD154 (CD40 ligand (CD40L)) has been demonstrated to play an essential role in the development of humoral and cellular immunity through its interaction with CD40. While earlier studies have examined the regulation of CD154 expression by transcriptional and posttranslational pathways, scant data exist on its regulation at a posttranscriptional level. In this report we demonstrate that CD154 mRNA is rapidly turned over in primary culture of activated human T lymphocytes. Moreover, we demonstrate that CD154 mRNA is unstable, but can be stabilized by treatment with either phorbol esters or calcium ionophores. To address this lability of CD154 mRNA, we examined the ability of cytoplasmic proteins to bind to its 3' untranslated region (3'UTR). Two major proteins (p25 and p50) capable of binding the 3'UTR of CD154 were identified. The p25 binding activity was associated with polysomes and appeared to correlate with CD154 mRNA instability. Intriguingly, these proteins did not appear to bind to the AU-rich elements present in the 3'UTR of CD154. Rather, their binding was localized to unique sites between nt 471-811 of the 3'UTR, which lack any classical AU-rich elements. These data suggest that these proteins interact with distinct cis-acting elements that are important in the posttranscriptional regulation of CD154 expression. As such, identifying these proteins will help us understand the signals that are necessary for CD154 expression by activated T cells.

Regulation of human monocyte HLA-DR and CD4 antigen expression, and antigen presentation by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2-D) has been shown to be a macrophage-derived cytokine, capable of regulating myeloid differentiation and T-cell activation in vitro. Therefore, we examined the effects of 1,25(OH)2-D on the monocyte phenotype and function of human peripheral blood monocytes as an index of its biologic role at an inflammatory site. 1,25(OH)2-D treatment consistently and specifically reduced HLA-DR and CD4 expression by monocytes, while CD14 and class I HLA antigen expression were unaffected. Expression of Fc gamma R I-III on monocytes was variably modulated by 1,25(OH)2-D treatment, but no differences in antibody-dependent cell cytotoxicity (ADCC) were observed, measured using either ADCC or anti-Fc gamma R-antibody expressing hybridomas. In contrast, the ability of monocytes to induce antigen-dependent T-cell proliferation was markedly reduced by 1,25(OH)2-D pretreatment for as little as 6 hours. Addition of interleukin-1 (IL-1), IL-6, or indomethacin did not restore antigen-dependent T-cell proliferation, suggesting that this observation was not secondary to changes in IL-1, IL-6, or PGE2 production induced by 1,25(OH)2-D. These data suggest that 1,25(OH)2-D treatment specifically modulates human monocyte phenotype and function, altering HLA-DR antigen expression and antigen presentation, while leaving lytic function intact. These findings may be relevant to the immunobiologic role of 1,25(OH)2-D.

1,25-Dihydroxyvitamin D3 modulates the effects of interleukin 2 independent of IL-2 receptor binding.

Previous studies have shown that 1,25-dihydroxyvitamin D3 (calcitriol) is a macrophage-derived cytokine and a potent inhibitor of IL-2 and interferon-gamma (IFN-gamma) production and T lymphocyte proliferation. The growth inhibitory effect of calcitriol is only partially reversed by IL-2 addition, suggesting IL-2 independent effects. In this report we characterize the IL-2-independent effects of calcitriol on lymphocyte activation. Calcitriol inhibited cellular transition from early to late G1 (G1A-G1B transition) in both the absence and presence of IL-2. Exogenous IL-2 did not increase either IFN-gamma production or transferrin receptor (TfR) expression in the presence of calcitriol despite increases in cell entry into late G1 and proliferation. Calcitriol treatment reduced TfR expression by activated T lymphocytes independent of their location in the cell cycle, further suggesting its independence from IL-2-mediated events. Combinations of rIL-2 and rIL-4 did not reverse calcitriol-dependent inhibition of proliferation and TfR expression to any greater degree than rIL-2 alone. Northern blot analysis demonstrated the decrease in IFN-gamma and TfR mRNA accumulation with calcitriol treatment was unaffected by exogenous IL-2. In contrast, IL-2R mRNA and protein were increased by IL-2, with superinduction in the presence of calcitriol, demonstrating that the lack of effect on IFN-gamma and TfR was not due to IL-2 insensitivity. Moreover, equivalent numbers of high-affinity IL-2R were expressed by both control and calcitriol-treated T lymphoblasts. Thus, lectin-activated T lymphocyte responsiveness to IL-2, as measured by IL-2R expression and proliferation, can be partly to completely dissociated from IFN-gamma production and TfR expression in the presence of calcitriol. Finally, IL-2-induced proliferation of unstimulated mononuclear cells and purified T lymphocytes was inhibited by calcitriol. These data indicate that local production of calcitriol by activated macrophages is capable of regulating T lymphocyte activation not only through suppression of IL-2 production, but also through additional mechanism(s), that are mediated at a post-IL-2R level.

Combined application of in vivo UV-crosslinking and in vitro label transfer in the examination of AU-rich sequence binding protein-RNA interactions.

A combination of in vivo UV light-induced crosslinking of nucleic acids to proteins and in vitro label transfer assay was applied to investigate specific interactions between AU-rich sequences (ARE) in the 3' UTR of lymphokine mRNAs and cytoplasmic AU-rich sequence element binding proteins (AUBP) in normal human lymphoblasts and MLA 144 gibbon lymphoid tumor cells. We demonstrate that a pool of cytoplasmic AUBP can be effectively crosslinked to RNA in vivo, suggesting a close association of these proteins with ARE sequences in the cytoplasm. We also show that the UV-crosslinked AUBP pool is markedly reduced in malignantly transformed MLA 144 cells compared with normal lymphoblasts, indicating weaker interaction between lymphokine ARE and AUBP in these tumor cells. Similar differences in AUBP-RNA associations were found between the membrane-bound polysomal subfractions of the two cell types where most of the AUBP activity was localized. We suggest that the decreased AUBP-mRNA association in MLA 144 cells might reflect a process concerned with disturbances of mRNA metabolism in the neoplastic phenotype.

Differentiation of a human monocytic cell line by 1,25-dihydroxyvitamin D3 (calcitriol): a morphologic, phenotypic, and functional analysis.

Several recent studies have suggested a role for 1,25-dihydroxyvitamin D3 (calcitriol) in myeloid differentiation. We have examined the effects of calcitriol on the U937 monoblast cell line and found that calcitriol, at near physiologic concentrations, is a potent inhibitor of U937 growth. Moreover, calcitriol induces differentiation to a monocyte-macrophage phenotype marked by enhanced alpha-naphthyl esterase staining. Morphologic changes were attended by de novo induction of the myeloid specific antigen detected by the monoclonal antibody AML-2-23, as well as dramatic increases in Fc receptors for IgG. In addition, calcitriol induced U937 cells to perform phagocytosis and antibody-dependent cellular cytotoxicity. These results indicate the potential activity of calcitriol in myeloid differentiation and additionally suggest a role for calcitriol in monocyte-macrophage activation.

Comparison of the effects of 1,25-dihydroxyvitamin D3 on T lymphocyte subpopulations.

Previous studies have demonstrated that 1,25-dihydroxyvitamin D3 (calcitriol) is a potent inhibitor of human T lymphocyte proliferation. It has been reported that only CD4+ cells are sensitive to the anti-proliferative action of calcitriol. To further evaluate this observation, we first performed cell cycle analysis of unfractionated phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) in the absence or presence of calcitriol. The CD4/CD8 ratio was similar between control and treated cells for each phase of cycle (G0----G1, S, G2 + M), suggesting that calcitriol did not selectively block proliferation of either T cell subpopulation. Secondly, the growth-inhibitory activity of calcitriol on PBMC selectively depleted of either CD4+ or CD8+ cells was comparable to that observed with unfractionated PBMC. Furthermore, the induction of transferrin receptors was inhibited by calcitriol to a comparable degree in each T cell subset, suggesting that equivalent inhibition of transition into late G1 was observed. Finally, calcitriol inhibited the proliferation of highly purified T cell subsets (greater than 99% pure) to equivalent degrees. These data suggest that T cell subsets defined by either the CD4 or by the CD8 antigen are both sensitive to the growth inhibitory effects of calcitriol.