Jack Silver

Resistance to Endotoxin Shock and Reduced Dissemination of Gram-Negative Bacteria in CD14-Deficient Mice

Endotoxin shock is the result of activation of the immune system by endotoxin/LPS, a component of Gram-negative bacteria. CD14, a GPI-anchored glycoprotein expressed strongly by monocyte/macrophages, is one of several receptors for endotoxin/LPS. The role of CD14 in bacterial-induced and LPS-induced shock was tested in CD14-deficient mice produced by gene targeting in embryonic stem cells. CD14-deficient mice were found to be highly resistant to shock induced by either live Gram-negative bacteria or LPS; however, at very high concentrations of LPS or bacteria, responses through non-CD14 receptors could be detected. Surprisingly, CD14-deficient mice also showed dramatically reduced levels of bacteremia, suggesting an unexpected role for CD14 in the dissemination of Gram-negative bacteria.

Overexpressed tau protein in cultured cells is phosphorylated without formation of PHF: implication of phosphoprotein phosphatase involvement

Pyramidal neurons in affected regions of Alzheimers disease (AD) brain contain neurofibrillary tangles (NFT), aggregates of paired helical filaments (PHF) composed mainly of phosphorylated microtubule-associated protein tau. To explore the role of tau phosphorylation in the aggregation of tau into PHF, we constructed mammalian cell culture systems producing high levels of intracellular phosphorylated tau. COS-1 fibroblast-like cells were transiently transfected to simultaneously express tau, MAP kinase (MAPK), and MAP kinase kinase (MAPKK), or alternatively to express tau and glycogen synthase kinase 3 (GSK3). B103 neuron-like cells (which contain MAPK but little tau or GSK3) were stably transfected to express tau or tau and GSK3. In both systems, GSK3-transfected cells contained tau AT8/M (defined by AT8 staining and tau PHF-like mobility), but MAPK-transfected cells required phosphatase inhibitors, such as okadaic acid (OKA) or calyculin (CAL), to produce tau AT8/M. In vitro, the same concentrations of CAL and OKA inhibit phosphatases 1 and 2A (PP1 and PP2A), except that 100-1000 times as much OKA is needed to inhibit PP1. Inducing tau phosphorylation at the AT8 site in MAPK-transfected cells required 2-10 times more OKA than CAL, suggesting both PP1 and PP2A helped block the phosphorylation. Though levels of tau AT8/M reached 2-8% of total cellular proteins in COS-1 cells, the ratio of particulate to supernatant tau levels did not increase, and no tangles were observed; perhaps post-translational modifications or co-aggregating proteins are needed to induce PHF.

Evidence for an altered T-cell receptor repertoire in Crohn's disease.

We have compared the frequencies of T cells expressing each of four different T cell receptor (TCR) V beta segments in lamina propria and peripheral blood lymphocytes of 12 Crohns disease (CD), six ulcerative colitis (UC), and 10 control patients in an attempt to identify disease-specific changes. The frequencies of CD4+ and CD8+ cells reacting with each of four fluoresceinated TCR-specific monoclonal antibodies directed against V beta 5, V beta 6.7a, V beta 8, and V beta 12 were determined by flow cytometry. There was no difference among the groups in the average frequency of any single V beta segment in either the CD4+ or CD8+ subpopulations. However, when the sum of the differences in V beta frequencies (delta score) between peripheral blood lymphocytes (PBL) and lamina propria lymphocytes (LPL) were determined for each individual, significant differences were observed between the CD4+ and CD8+ populations and among the patient groups. In all three patient groups, there were significant individual differences between LPL and PBL in the frequencies of CD8+ and CD4+ cells reacting with the four V beta-specific mAb. In Controls and UC, this difference was, on average, two-fold greater in CD8+ cells than in CD4+. In CD, however, this difference was, on average, the same for CD8+ and CD4+ cells. These observations suggest that (1) the human colonic LPL TCR repertoire is normally different from that of PBL, especially in the CD8+ population and (2) there is an alteration in the LPL TCR repertoire in CD which is not observed in Controls or UC.

Vβ‐Specific Activation of T Cells by the HIV Glycoprotein gp 160

Studies by several groups have suggested that HIV infection in vivo results in a Vβ‐specific alteration of the TCR repertoire and that this might play a role in the pathogenesis of AIDS. However, there is very little agreement as to which Vβ segments are affected. In order to circumvent the confounding factors present in vivo we have examined the abilities of both a crude protein extract of HIV and purified gp 160 to alter the Vβ repertoire of normal T cells in vitro. We find that both a crude extract of HIV as well as gp 160 specifically activate T cells expressing a common set of Vβ segments (Vβ3, 12, 14, 15, and sometimes Vβ17 and 20) in individuals of disparate HLA type. This set of Vβ segments is remarkably similar to those recognized by staphlococcal enterotoxin B and supports the hypothesis that bacterial superantigens produced by opportunistically acquired micro‐organisms could have an exacerbating effect in AIDS.

CD4+ Cell Oligoclonality in Crohn's Disease: Evidence for an Antigen-Specific Response

To identify disease-specific T cell changes that occur in Crohns disease (CD) the T-cell receptor (TCR) BV repertoires of lamina propria lymphocytes (LPL) from both disease-active and disease-inactive colonic tissue of three CD patients were compared by a quantitative polymerase chain reaction (qPCR) and CDR3 length analysis. It was observed that the BV repertoires of LPL isolated from the disease-active and disease-inactive parts of the colon of the same individual were different, and most of the differences occurred in CD4+ LPL with very few differences in the CD8+ populations of LPL. Although the pattern of BV segments that was increased in disease-active relative to disease-inactive tissue was different for all three CD patients, there was an increase in the levels of BV11, 13S2, 15, 16, and 17 segments in the disease-active tissue of all three patients. Standard CDR3 length analysis of BV11, 13S2, 15, 16, and 17 segments revealed that in two of the three CD patients there was a striking degree of TCR oligoclonality in the disease-active tissue that was absent from disease-inactive tissue of the same individual. Additional differences between the disease-active and disease-inactive tissues were observed using a more refined method of CDR3 length analysis, which employs BV- and BJ-specific primers. These observations suggest that at least some of the inflammation in CD is the result of responses by CD4+ T cells to specific antigens.

Analysis of the peripheral blood t-cell receptor (tcr) repertoire in monozygotic twins discordant for crohn's disease

T cell involvement in the inflammatory process of Crohns Disease (CD) is evident by an increase in activated T cells and their cytokines in actively inflamed CD tissue. It has been suggested that CD may involve a superantigen based on the observation that a significant proportion of CD patients express elevated levels of V beta 8+ T cells in their peripheral blood compared to normal controls. In order to determine whether a superantigen might play a role in the pathogenesis of CD we have compared the TCR repertoires of four pairs of monozygotic twins discordant for CD. By using monozygotic twins, we could rule out the effects of HLA and other genes on the TCR repertoire. The TCR repertoires were analyzed by using a panel of V-segment-specific mAb and by quantitative polymerase chain reaction (qPCR) using V beta-specific oligonucleotide primers. In all cases the TCR repertoires of the affected and unaffected sibs were strikingly similar. We did not observe any TCR segment that was consistently altered in frequency or expression levels in all of the affected sibs compared to their identical twin. Furthermore, we did not see an increase in V beta 8+T cells in the peripheral blood of the CD sibs relative to their normal counterpart. These studies suggest that the presence of CD does not alter the TCR repertoire of peripheral blood in any obvious way and argue against the role of a superantigen in the etiology of pathogenesis of CD.

Reduced bacterial dissemination and liver injury in CD14-deficient mice following a chronic abscess-forming peritonitis induced by Bacteroides fragilis

Abstract The CD14 myelomonocytic differentiation antigen plays a major role in acute Gram-negative infections with Escherichia coli; however, its role in chronic infections has not yet been analyzed. To address this question, we studied the role of CD14 in a chronic abscess-forming peritonitis, induced by Bacteroides fragilis. B. fragilis (3×108 CFU/ml) were resuspended in a liquid nutrient agar and injected into the peritoneal cavity of CD14-deficient (CD14–/–) and normal C57BL/6J (CD14+/+) mice, respectively. After 3 days there was a severe phlegmonous intra-abdominal inflammation in both groups. After 7 days an abscess-forming peritonitis developed and by 14 days the infectious foci were compartimentalized. These obser-vations were indistinguishable between CD14–/– and CD14+/+ mice. Although no differences were seen in abscess formation, CD14–/– mice were able to clear B. fra-gilis more efficiently from the blood than CD14+/+ mice. After 3, 7, and 14 days blood cultures were B. fragilis positive in 11% (1/9), 20% (2/10), and 0% (0/9) in CD14–/– compared with 90% (9/10), 78% (7/9), and 20% (2/10) in CD14+/+ mice, respectively (P<0.05). Furthermore, although the infection resulted in hepatocellular necrosis and severe hepatitis in both groups, at day 14 the liver cell damage was more severe in CD14+/+ than in CD14–/– mice (P<0.05). These results show that the chronic abscess formation induced by B. fragilis capsular polysaccharides is CD14 independent; however, bacterial clearance and/or dissemination and liver cell damage are at least partially influenced by CD14-dependent mechanisms.

Evidence that the Receptor for Soluble CD14:LPS Complexes may not be the Putative Signal‐Transducing Molecule Associated with Membrane‐Bound CD14

Membrane‐bound CD14 acts as a receptor for lipopolysaccharide (LPS) on monocytes/macrophages and neutrophils. Studies have suggested that the activation of monocytes/macrophages by the binding of LPS to membrane‐bound CD14 may require the association of a signal‐transducing molecule with membrane‐bound CD14. The observation that non‐CD14 expressing cells, such as endothelial cells, can nevertheless be activated by a complex of LPS and a soluble form of CD14 (sCD14) suggests that the receptor for this complex may be identical to the signal transducing molecule associated with membrane‐bound CD14. The studies described show that two CD14‐specific MoAb are able to block the LPS‐induced activation of endothelial cells but do not affect the response of monocytes to LPS. This suggests that the interaction of the sCD14:LPS complex with endothelial cells is distinct from the interaction of membrane‐bound CD14 with its putative signal‐transducing molecule.

THE INFLUENCE OF NON-HLA GENES ON THE HUMAN T-CELL RECEPTOR REPERTOIRE

We previously demonstrated a central role for HLA genes in determining the T‐cell receptor (TCR) repertoire. However, these studies also suggested that other genetic factors might also play a role in the development of this repertoire. In order to assess the role of non‐HLA genes in the development of the TCR repertoire, we have analysed and compared the TCR repertoires of individuals in three families consisting of both monozygotic twins as well as an HLA‐identical sib. TCR repertoire analysis was performed with both V‐segment‐specific MoAb and the polymerase chain reaction using TCRBV segment‐specific oligonucleotide primers. We observed that in every case the TCR repertoires of identical twins were more similar to each other than to their HLA‐identical sib. Furthermore, in one family we were able to show by genotype analysis that most of the differences in repertoire between the identical twins and their HLA‐identical sib were caused by polymorphisms in the TCR genes that influence expression levels. These studies document an important role for non‐HLA genes in determining the TCR repertoire in man and raise the possibility that such TCR polymorphisms may play a signiflcant role in determining disease susceptibility.

Marked γδ T-Cell Decrease in Peripheral Blood of Patients with Primary Biliary Cirrhosis (PBC)

PBC is a cholestatic liver disease of unknown etiology with autoimmune features that is often associated with other autoimmune diseases. We analyzed peripheral blood T-cell subsets in patients groups with PBC (n = 11), non-PBC hepatobiliary disease (n = 11) and an age and sex matched control group (n = 11) by two color FACS-analysis. Seven out of eleven PBC patients exhibited markedly lowered and nearly undetectable levels of y8 T-cells (< 0.8%). None of the individuals in the non-PBC hepatobiliary disease (HBD) gkroup or the normal control group had y8 values below 1%. The other four individuals in the PBC group had γδ values within the normal range. Overall, the PBC group had a statistically significant, lowered mean percentage of γδ T-cells (L.50%) as compared to the hepatobiliary disease group (3.76%) and the control group (4.22%, p = 0.01). The percentages of CD4+ and CD8 + and αβ TCR + CD4-CD8 − double negative cells in PBC patients did not differ from the control group. PBC patients with normal γδ ...