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Biomedical subjects

P E Lipsky

Publications and source records attributed to P E Lipsky.

At least 19 recordsLinked to original sources
PubMed1995

Elevated Th1- or Th0-like cytokine mRNA in peripheral circulation of patients with rheumatoid arthritis. Modulation by treatment with anti-ICAM-1 correlates with clinical benefit.

The current studies examined whether cytokine patterns indicative of an imbalance in Th1 and Th2 cells could be identified in PBMC of patients with active rheumatoid arthritis (RA). To investigate this possibility, a reproducible PCR technique to assess cytokine mRNA levels in PBMC was employed that minimized in vitro manipulation of the cells. Seven of 14 RA patients had increased mRNA levels for IL-2, 5/14 for IFN-gamma, 3/14 for IL-4, and 4/14 for the IL-2R alpha-chain, compared with normal donors. Whereas 4 patients had elevated mRNA for IL-2 and IFN-gamma, indicative of an increase in activated Th1 or Th0 cells, 1 of 14 patients expressed low levels of IL-2 and IFN-gamma and high levels of IL-4 mRNA. Seven RA patients were treated with a mAb to ICAM-1 (CD54). To determine whether changes in cytokine mRNA levels might be associated with and/or account for the anti-inflammatory effect of anti-ICAM-1 mAb therapy, changes in cytokine mRNA levels were assessed and correlated with clinical improvement. Anti-ICAM-1 mAb administration was followed by a prompt and transient increase of IFN-gamma mRNA. Elevation of IFN-gamma mRNA expression throughout the treatment period reflected a temporary increase in the number of circulating CD3+CD4+ T cells, suggestive of altered circulatory patterns of activated Th1-like cells and was related to clinical efficacy. The results indicate that elevated cytokine mRNA levels characteristic for Th1 cells can be detected in the PBMC in active RA and, furthermore, that anti-ICAM-1 mAb may be beneficial in RA by altering the recruitment of activated Th1-like cells into the synovium. This assumption further strengthens the hypothesis of a significant contribution of Th1-like cells to the pathogenesis of RA.

Antibodies↗
PubMed1995

Regulation of human B cell function by recombinant CD40 ligand and other TNF-related ligands.

To assess the potential of CD40 ligand (CD40L) and the related molecules CD27 ligand (CD27), CD30 ligand (CD30L), and membrane TNF-alpha to stimulate B cell responses, expression of these proteins in the baculovirus system was performed. Sf9 cells expressing these membrane molecules were cultured with normal human B cells and a variety of B cell lines to assess the functional outcome. The signal provided by CD40L promotes aggregation of B cells, stimulates vigorous proliferation, and induces germ-line transcription of downstream heavy chain constant region genes in the absence of cytokine costimulation. In contrast, CD27L, CD30L, and TNF-alpha had no effects on B cell proliferation. CD27L and TNF-alpha had no effect on the induction of germ-line transcripts, whereas CD30L consistently inhibited constitutive and CD40L-induced germ-line transcription of the epsilon gene by B cell lines that express CD30. These results demonstrate the various members of the TNF family exert specific effects on human B cell function, with CD40L and CD30L providing powerful, but opposing, effects on l epsilon transcription.

Antigens, CD↗
PubMed1995

Protection from T helper cell-mediated graft-versus-host disease by the presence of an MHC class I alloantigen is associated with perturbation of MHC class II-restricted responses by class I-derived peptides.

Lethal graft-vs-host disease (GVHD) develops after transfer of CTL-depleted spleen and bone marrow cells from C57BL/6 (B6) mice to irradiated MHC class II disparate (B6xbm12)F1 recipients. Onset of lethal GVHD is significantly delayed in (bm1xbm12)F1 recipients of the same donor inoculum despite the additional MHC class I disparity (H-2Kbm1). To investigate the basis of this protective effect, hybridomas were generated from T cells activated in vivo during GVHD in both strain combinations. T cells from B6-->(B6xbm12)F1 mice generated a significantly higher frequency of B6.C-H-2bm12 (bm12)-specific T hybridomas (110/178, 62%) than T cells from B6-->(bm1xbm12)F1 mice (102/218, 47%). Some bm12-specific T hydridomas exhibited lesser responses to (bm1xbm12)F1 than to (B6xbm12)F1 APC. Moreover, bm1 peptides spanning a 14-amino acid region that includes the three amino acids that differ from H-2Kb were found to inhibit responses of some bm12-specific T hybridomas and to decrease significantly responses of splenic B6 CD4+ T cells to bm12 APC. Notably, the frequency of bm12-reactive hybridomas susceptible to inhibition by bm1 peptides generated from B6-->(B6xbm12)F1 mice was significantly greater than that generated from B6-->(bm1xbm12)F1 mice. Additional analysis using L cell transfectants indicated that hybridomas responding to the bm12 mutation at position 70 alone were rarely inhibited by bm1 peptides. The data indicate that expression of bm1-derived peptides can influence the frequency and specificity of alloreactive CD4+ T cells stimulated in vivo and thus may alter the course of GVHD.

Amino Acid Sequence↗
PubMed1995

Expression of CD45RB and CD27 identifies subsets of CD4+ memory T cells with different capacities to induce B cell differentiation.

The capacity of four subsets of CD4+ memory T cells, defined by expression of CD45RB and CD27, to provide help for B cells was examined. Larger amounts of Ig were induced by CD45RBdimCD27- cells compared with the CD45RBdimCD27+ population, whereas CD45RBbrightCD27+ or CD27- cells were poor inducers of Ig synthesis. Mitomycin C treatment, which prevents suppressive activity, markedly enhanced Ig production supported by each subset except for CD45RBbrightCD27- cells. Mitomycin C-treated CD45RBdim cells remained the most efficient inducers of Ig production, but no difference was detected between CD27+ and CD27- cells. The subsets also differed in their ability to proliferate and secrete cytokines, but these differences did not explain variations in the capacity to provide help for B cells. Both CD27- subsets exhibited decreased proliferation and uniquely secreted IL-4, with the CD45RBdimCD27- subset producing the greatest quantities of IL-4. No differences in IL-2 and IFN-gamma production were found. IL-10 secretion increased with the acquisition of the CD45RBdim phenotype and, within the CD45RBdim cells, with the loss of CD27. Staining for cytoplasmic cytokines indicated that individual populations of CD27-CD4+ helper T cells produced either IL-4 or IFN-gamma, whereas more than half of the IL-4 producers also synthesized IL-2. Finally, the different abilities of CD4+ memory T cell subsets to support B cell differentiation did not relate to variations in the expression of CD40 ligand. These results indicate that within the CD4+ memory T cell population an increase of helper activity associates with the shift from a CD45RBbright to a CD45RBdim phenotype. Within the CD45RBdim subset, the loss of CD27 is associated with a reduction of suppressive activity.

Adult↗
PubMed1995

Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction.

The VH gene repertoire of human peripheral B cells was analyzed using PCR analysis of individual blood B cells. Because genomic DNA of single B cells was analyzed, data from both productive and nonproductive VDJ rearrangements were obtained. Nine out of 75 B cells contained both functional and nonfunctional rearrangement products, whereas 62/75 had a single productive VDJ rearrangement. The distribution of VH families was ordered in accordance with the germline complexity, although a bias toward VH3 and some of its members was found. This bias was noted in both the productively and nonproductively rearranged repertoires, indicating that it resulted from molecular and not selective processes. Evidence for negative selection of certain VH3 and VH4 family members was noted in that they were found less often as productive than nonproductive VDJ rearrangements. In addition, evidence for positive selection based on CDR3 was obtained, in that JH6 and DXP'1 were found at a higher frequency in the productively compared with the nonproductively rearranged repertoire. The CDR3 segments were markedly heterogenous, with a much greater degree of variability noted in the nonproductive VDJ rearrangements. Finally, a much greater frequency of mutations was noted in the nonproductively rearranged VH genes within individual B cells. These results have begun to delineate the human peripheral B cell repertoire and identify processes that shape it both positively and negatively.

B-Lymphocytes↗
PubMed1995

The CD40 ligand expressed by human B cells costimulates B cell responses.

The possibility that activated B cells might express a ligand for CD40 that was of functional importance for B cell responses was examined by using highly purified human peripheral blood B cells, as well as a variety of B lymphoblastoid cell lines and hybridomas. Following stimulation with the combination of a calcium ionophore and a phorbol ester, human B cells bound a soluble fusion protein containing the extracellular portion of CD40 and the Fc region of IgG1 (CD40.Ig). A variety of B cell lines and hybridomas also bound CD40.Ig, either constitutively or after activation. In addition, CD40.Ig specifically immunoprecipitated a 33-kDa glycoprotein from surface 125I-labeled activated B cells. The nucleotide sequence of the coding region of the CD40 ligand mRNA amplified by RT-PCR from activated T cells and B cell lines was identical. The CD40 ligand expressed on human B cells was important functionally because homotypic aggregation of CD40 ligand-expressing B cells was inhibited by the CD40.Ig construct. Additionally, RNA and DNA synthesis as well as Ig production by polyclonally activated, highly purified peripheral B cells and a variety of B cell lines were inhibited significantly by the CD40.Ig construct. Finally, B cell lines expressing the CD40 ligand induced Ig production from resting normal B cells in a CD40-dependent manner. These results indicate that human B cells express a ligand for CD40 that is identical with that expressed by activated T cells and that the B cell-expressed CD40 ligand plays an important role in facilitating responses of activated B cells.

Adult↗
PubMed1995

Suppression of the proliferation of Ras-transformed cells by fluoromevalonate, an inhibitor of mevalonate metabolism.

Mevalonate is the precursor of a number of different products potentially required for the growth of cells, including the prenylated oncoprotein Ras. To determine whether inhibition of mevalonate metabolism would selectively block proliferation of Ras-transformed cells, 6-fluoromevalonate (Fmev), an inhibitor of diphosphomevalonate decarboxylase, was used to block the synthesis of prenyl-derived lipids and prenylated proteins in interleukin-3 (IL-3)-dependent FDC-P1 cells (control FDC-P1 cells) and FDC-P1 cells transformed with oncogenic Ras (RasDC cells) that proliferated in the absence of IL-3. Fmev completely inhibited synthesis of prenyl-derived lipids and prenylated proteins and blocked proliferation of FDC-P1 and RasDC cells. Restoration of the proliferation of Fmev-blocked FDC-P1 cells required both an exogenous source of cholesterol and prevention of the accumulation of mevalonate and the mevalonate phosphates with lovastatin. In contrast, ongoing IL-3-independent proliferation of Fmev-blocked RasDC cells was not completely restored by providing exogenous cholesterol and preventing the accumulation of inhibitory mevalonate product(s). However, these cells proliferated when cultures were supplemented with IL-3 together with exogenous cholesterol and lovastatin, implying that Fmev had prevented Ras-dependent, IL-3-independent growth. Fmev markedly diminished total cellular Ras in RasDC cells. In contrast, lovastatin depleted membrane-associated Ras and increased cytosolic Ras but did not diminish total cellular Ras. These data indicate that Fmev depletes total cellular Ras and specifically inhibits the autonomous growth of Ras-transformed cells.

Animals↗
PubMed1995

Phenotypic characterization of CD4+ T cells that exhibit a transendothelial migratory capacity.

The phenotype of CD4+ T cells capable of transendothelial migration was determined using an in vitro model system, in which cells migrate through a monolayer of endothelial cells (EC) on collagen gels. A specific subset of resting CD4+ memory T cells was found to migrate. T cells within this subset can be defined by the bright expression of CD11a, CD26, CD44, and CD49d. Additionally, the migratory CD4+ T cell population is largely CD58bright, CD31-, CD62L-, and is also enriched in cells that brightly express CD49c, CD49e, and CD49f. Only a minority of the cells are activated, as indicated by expression of CD69. The EC were found to play a central role in facilitating migration of this subset because selective enrichment of CD11abright, CD26bright, CD44bright, CD4+ T cells was not observed when cells migrated in the absence of EC. Activation of the T cells induced a modest degree of migration of an additional subset of CD45RA+, CD31+ naive T cells. In contrast, TNF-alpha activation of the EC increased the transendothelial migration of an additional subset of activated memory T cells that expressed CD69 and CD62L. Neither activation of the T cells, stimulation of the EC, nor the presence of macrophage inflammatory protein-1 alpha (MIP-1 alpha) or RANTES, however, altered the phenotype of the majority of the migratory CD4+ T cell population, which is characteristic of a particular stage of memory cell differentiation. These results suggest that CD4+ T cells acquire the capacity for transendothelial migration at a specific phase of maturation that is only minimally altered by the activation of either the T cell or the EC, or by the presence of specific chemokines in the subendothelial matrix.

Antigens, CD↗
PubMed1995

Induction of persistent T cell hyporesponsiveness in vivo by monoclonal antibody to ICAM-1 in patients with rheumatoid arthritis.

Rheumatoid arthritis patients undergoing treatment with a murine mAb (BIRR1) to ICAM-1 were studied to determine the effects of the treatment on T cell responsiveness assayed in vitro. Previous studies had demonstrated that over the 5-day treatment period, there was a transient increase in circulating T cells that returned to base line 3 days after therapy. The transient lymphocytosis correlated with a loss in delayed-type hypersensitivity reactivity during the time of Ab administration. However, neither the increase in T cell numbers nor the inhibition of delayed-type hypersensitivity responses correlated with the immediate clinical benefit of therapy or the prolonged nature of the response to therapy in some patients. The current studies show that after the return of lymphocyte numbers to pretreatment levels, a decrease in T lymphocyte responses to suboptimal activation signals, including accessory cell-dependent (low dose PHA and soluble anti-CD3 mAb) and accessory cell-independent stimuli (immobilized anti-CD3 mAb), was observed. However, responses to recall Ags were preserved. Depressed T cell responses were not the result of diminished accessory cell function or production of suppressive factors by monocytes, but rather reflected decreased IL-2 production. The duration of T cell hyporesponsiveness was variable in length but lasted up to 5 mo after treatment with anti-ICAM-1 mAb. The induction and persistence of T cell hyporesponsiveness correlated with an improvement in disease activity in treated patients. These studies show that treatment with anti-ICAM-1 mAb can induce T cell hyporesponsiveness that correlates with and may explain sustained therapeutic benefit in patients with rheumatoid arthritis.

Antibodies, Monoclonal↗
PubMed1995

Elevated interleukin-10 levels in patients with rheumatoid arthritis.

OBJECTIVE: Interleukin-10 (IL-10) has been shown to exert both antiinflammatory and immunostimulatory effects in vivo and in vitro. We therefore sought to examine the role of this cytokine in rheumatoid arthritis (RA) by assessing serum and synovial fluid IL-10 levels. METHODS: Serum and synovial fluid samples were collected from patients with RA and patients with various inflammatory, infectious, and noninflammatory arthritides (controls). IL-10 was assayed using an IL-10-specific enzyme-linked immunosorbent assay, and messenger RNA (mRNA) levels were assessed by semi-quantitative polymerase chain reaction (PCR) techniques. RESULTS: Both RA serum and synovial fluid contained significantly elevated IL-10 levels compared with levels in normal subjects or in control patients (P < 0.01). Some patients with spondylarthropathy also manifested increased serum levels of IL-10. Serum levels of IL-10 did not correlate with standard measures of clinical activity, but were shown to correlate significantly with serum rheumatoid factor (RF) titers and in vitro levels of spontaneous IgM-RF production (P < 0.05). PCR analyses demonstrated the constitutive expression of IL-10 mRNA by the non-T cell population, and semiquantitative PCR analysis documented elevated levels of IL-10 mRNA in circulating mononuclear cells of those RA patients who were not treated with slow-acting antirheumatic drugs. Analysis of IL-10 mRNA revealed the cytokine to be of human, and not viral, origin. CONCLUSION: These data suggest that there is increased production of IL-10 by non-T cells in patients with RA. This may contribute to the diminished T cell function and increased antibody and RF production in these patients.

Adult↗
PubMed1995

Evidence for a CD40 response element, distinct from the IL-4 response element, in the germline epsilon promoter.

Engagement of CD40 by its ligand induces transcription of unrearranged Ig heavy chain genes, an initial step in switch recombination. The following studies were undertaken to understand the molecular basis of this response. Co-culture of S19 cells expressing membrane-bound CD40 ligand (CD40L) encoded by recombinant baculovirus with EBV-transformed B cell lines induced germline transcription of the epsilon gene in the absence of cytokines. To identify a putative CD40 response element, a reporter construct consisting of the 777 bp of the 5' flank of the human l epsilon region linked to the chloramphenicol acetyl transferase (CAT) gene was stably transfected into B cell lines. Stimulation with either CD40L-expressing Sf9 cells or IL-4 induced CAT activity. Deletional analysis of this promoter region confirmed that an IL-4 response element was identified within a 63 bp segment 3' to the IL-4-responsive element that was responsive to CD40 ligation. These results indicate that the germline epsilon promoter contains a CD40 response element that is distinct from that accounting fro IL-4 responsiveness. Activity of this response element may explain the capacity of ligation of CD40 to induce germline epsilon transcripts in the absence of cytokines.

Animals↗
PubMed1995

Alterations in leukocyte adhesion molecule expression after burn injury.

OBJECTIVE: To determine if thermal injury alters the expression of leukocyte adhesion molecules. DESIGN: This is a controlled experimental animal study. MATERIALS AND METHODS: Partial thickness burns were created on the backs of New Zealand White rabbits. At 30 minutes, 2 hours, 4 hours, and 24 hours after burn, skin was harvested for immunohistochemistry. Monoclonal antibodies were used to study changes in intercellular adhesion molecule 1 (ICAM-1), E-selectin, and leukocyte CD11a. Staining was graded on a scale of 0 to 4. MEASUREMENTS AND MAIN RESULTS: ICAM-1 was significantly decreased at 24 hours after burn (p < 0.007, Wilcoxon signed rank test). CD11a was increased at 30 minutes (p < 0.02), 2 hours (p < 0.02), and 24 hours (p < 0.006). E-selectin was increased at 2 hours (p < 0.03). CONCLUSION: Thermal injury alters the expression of leukocyte adhesion molecules.

Animals↗
PubMed1995

The identity of immunosuppressive components of the ethyl acetate extract and chloroform methanol extract (T2) of Tripterygium wilfordii Hook. F.

A variety of preparations of Tripterygium wilfordii Hook.F (TWHF) have been reported to be effective in the treatment of autoimmune diseases, including a chloroform methanol extract termed T2 and an ethyl acetate (EA) extract. The immunosuppressive activity of the EA extract was analyzed and the components accounting for this effect determined and compared to those of T2. More than 0.25 microgram/ml of the EA extract inhibited antigen- and mitogen-stimulated human T cell proliferation. The inhibitory effect of the EA extract on T cell proliferation resulted largely from suppression of interleukin-2 production. At concentrations that inhibited T cell function, the EA extract also profoundly suppressed [3H]-thymidine incorporation by mitogen-stimulated B cells, but it did not inhibit antigen presentation by monocytes and only modestly affected interleukin-6 production by lipopolysaccharide-stimulated monocytes. The profile of inhibition was comparable to that previously reported for the chloroform-methanol extract of Tripterygium wilfordii Hook.F, T2. To delineate the components of these extracts that might account for their immunosuppressive effect, we analyzed the composition of diterpenoid compounds. Both extracts contained triptolide and tripdiolide as the major immunosuppressive diterpenoids, but at different concentrations. Comparison of the composition of these extracts and the inhibitory capacity of the purified components indicated that the triptolide concentration of the EA extract can account for its immunosuppressive activity, although the combination of both triptolide and tripdiolide or other unknown components may be necessary to explain the inhibitory effects of T2.

Acetates↗
PubMed1994

Reactivity of hybridomas derived from T cells activated in vivo during graft-versus-host disease.

To examine the specificity of T helper cells activated during murine graft-vs-host disease (GVHD), T cell hybridomas from GVHD spleens and livers were generated and analyzed. CTL-depleted C57BL/6 (B6) donor cells were injected into irradiated (B6 x bm12)F1 or (bm1 x bm12)F1 recipient mice. Five or fourteen days later, cells from livers and spleens were fused directly with the TCR-deficient (alpha beta)- BW5147 thymoma line. The in vivo-activated T cells produced hybridomas as efficiently as either T cells activated in a primary mixed lymphocyte reaction or expanded in vitro after isolation from GVHD mice. Overall, 91% (396 of 437) of hybridomas generated from GVHD animals responded to immobilized anti-CD3 and 56% (220 of 396) of these hybridomas responded specifically to APC expressing host bm1 or bm12 alloantigens. More than 80% of bm12-specific hybridomas expressed CD4; all (53 of 53) of the bm12-specific hybridomas tested reacted to homozygous bm12 APC. Of the alloreactive T hybridomas generated from B6-->(bm1 x bm12)F1 GVHD mice, 7% responded to bm1 APC. Five bm1-specific hybridomas were analyzed further. One CD4+ hybridoma recognized a bm1 peptide presented by self I-Ab and was blocked by anti-Ia Ab; the other four hybridomas, two of which also expressed CD4, responded to transfected L cells expressing H-2Kbm1 and were not inhibited by anti-Ia Ab. These results indicate that a high percentage of CD4+ T hybridomas generated from freshly isolated T cells activated in vivo during GVHD are specific for host MHC class II or class I alloantigens.

Amino Acid Sequence↗
PubMed1994

Human peripheral blood dendritic cell subsets. Isolation and characterization of precursor and mature antigen-presenting cells.

Dendritic cells (DC) are the major APC capable of stimulating resting T cells in human peripheral blood (PB). Recent evidence suggested that various subsets of DC and monocytes might circulate in human PB, but their exact phenotype and function had not been delineated. We have previously characterized a population of human PB DC precursors that express the myeloid marker CD33, but not the monocyte marker CD14. To identify and characterize further functional myeloid APC subsets, triple color FACS analysis and sorting was used. A CD33dimCD14dimCD16+ monocyte subset, with similar APC function but less efficient accessory function than CD14bright monocytes, was isolated. In addition to the CD33+CD14dimCD16- DC precursors, a smaller population (0.1 to 0.2% of PBMC) of CD33brightCD14dimCD16- cells with potent APC function was identified. This DC population expressed greater amounts of MHC class II, adhesion, and accessory molecules, and demonstrated a greater costimulatory capacity when freshly isolated than CD33dimCD14dim DC precursors, and therefore had the characteristics of mature, possibly tissue-derived DC. When freshly isolated, however, these DC did not express B7, and up-regulation of accessory function occurred after in vitro differentiation. These data demonstrate multiple circulating myeloid accessory and APC subsets in human PB. Phenotypic and functional differences suggest that they are at different stages of differentiation, and have specialized roles in Ag presentation in vivo. Furthermore, full functional DC differentiation, associated with B7 expression and the capacity to activate T cells maximally, is likely to occur only in specific physiologic circumstances.

Antigens, CD↗
PubMed1994

Assessment of a positive selection technique using an avidin column to isolate human peripheral blood T cell subsets.

The current studies were designed to assess a new technique for positively selecting human T cells from whole peripheral blood mononuclear cells using the minimal amount of monoclonal antibody required to bind the T cell to an avidin column indirectly via a biotin-conjugated secondary antibody. Positive selection of T cells has previously been avoided because the saturating amounts of antibodies required for other isolation procedures can lead to aberrant results in assays of T cell activation and function. The avidin column technique for obtaining purified T cell subsets was compared to a multi-step procedure that included negative selection panning. The positive selection technique was easily performed within 4 h whereas the negative selection technique required a minimum of 12 h to complete. The avidin column technique proved to be a rapid and simple method for isolating T cell subsets of high purity and normal functional capabilities. Since minimal amounts of monoclonal antibodies were used for the purification protocol, no consistent inhibitory or stimulatory effect of the residual antibody was noted in assays of activation and proliferation of positively selected T cells compared to T cells isolated by negative selection panning.

Adult↗