Evidence for a functional role of the second C5a receptor C5L2

During experimental sepsis in rodents after cecal ligation and puncture (CLP), excessive C5a is generated, leading to interactions with C5aR, loss of innate immune functions of neutrophils, and lethality. In the current study, we have analyzed the expression of the second C5a receptor C5L2, the putative â defaultâ or nonsignaling receptor for C5a. Rat C5L2 was cloned, and antibody was developed to C5L2 protein. After CLP, blood neutrophils showed a reduction in C5aR followed by its restoration, while C5L2 levels gradually increased, accompanied by the appearance of mRNA for C5L2. mRNA for C5L2 increased in lung and liver during CLP. Substantially increased C5L2 protein (defined by binding of 125Iâ antiâ C5L2 IgG) occurred in lung, liver, heart, and kidney after CLP. With the use of serum ILâ 6 as a marker for sepsis, infusion of antiâ C5aR dramatically reduced serum ILâ 6 levels, while antiâ C5L2 caused a nearly fourfold increase in ILâ 6 when compared with CLP controls treated with normal IgG. When normal blood neutrophils were stimulated in vitro with LPS and C5a, the antibodies had similar effects on release of ILâ 6. These data provide the first evidence for a role for C5L2 in balancing the biological responses to C5a.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154410/1/fsb2fj043424fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154410/2/fsb2fj043424fje-sup-0040.pd

Protection of innate immunity by C5aR antagonist in septic mice

Innate immune functions are known to be compromised during sepsis, often with lethal consequences. There is also evidence in rats that sepsis is associated with excessive complement activation and generation of the potent anaphylatoxin C5a. In the presence of a cyclic peptide antagonist (C5aRa) to the C5a receptor (C5aR), the binding of murine 125Iâ C5a to murine neutrophils was reduced, the in vitro chemotactic responses of mouse neutrophils to mouse C5a were markedly diminished, the acquired defect in hydrogen peroxide (H2O2) production of C5aâ exposed neutrophils was reversed, and the lung permeability index (extravascular leakage of albumin) in mice after intrapulmonary deposition of IgG immune complexes was markedly diminished. Mice that developed sepsis after cecal ligation/puncture (CLP) and were treated with C5aRa had greatly improved survival rates. These data suggest that C5aRa interferes with neutrophil responses to C5a, preventing C5aâ induced compromise of innate immunity during sepsis, with greatly improved survival rates after CLP.â Huberâ Lang, M. S., Riedeman, N. C., Sarma, J. V., Younkin, E. M., McGuire, S. R., Laudes, I. J., Lu, K. T., Guo, R.â F., Neff, T. A., Padgaonkar, V. A., Lambris, J. D., Spruce, L., Mastellos, D., Zetoune, F. S., Ward, P. A. Protection of innate immunity by C5aR antagonist in septic mice. FASEB J. 16, 1567â 1574 (2002)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154360/1/fsb2fj020209com.pd

Fragile Site Instability in <i>Saccharomyces cerevisiae</i> Causes Loss of Heterozygosity by Mitotic Crossovers and Break-Induced Replication

<div><p>Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.</p></div

Blood mononuclear cell production of TNF‐α and IL‐8: engagement of different signal transduction pathways including the p42 MAP kinase pathway

Recent studies of human peripheral blood mononuclear cells (PBMC) stimulated with IgG subclasses have suggested that tumor necrosis factor α (TNF‐α) and interleukin‐8 (IL‐8) production proceed along different signal transduction pathways. To investigate this possibility, inhibitors of signal transduction pathways were employed. Human PBMC were pretreated with various inhibitors before being added to IgG2‐coated wells and 4‐h supernatant fluids evaluated for cytokine content. The effects of various inhibitors on MAP kinase activation were determined. Inhibitors of protein tyrosine kinases, phosphatases, and phospholipase C decreased TNF‐α and IL‐8 production, suggesting that all three enzyme pathways are involved in cytokine generation. Inhibitors of G‐proteins had differing effects: pertussis toxin inhibited IL‐8 but not TNF‐α production, whereas cholera toxin inhibited TNF‐α but not IL‐8 production. Pretreatment of PBMC with pertussis toxin resulted in reduced IgG2‐induced calcium mobilization, whereas cholera toxin had no effect, correlating with the effects of pertussis toxin on IL‐8 expression. Inhibitors of protein kinase C (PKC) completely blocked TNF‐α generation but had no effect on IL‐8 production. Gö6976, which inhibits certain isoforms of PKC, inhibited production of both IL‐8 and TNF‐α. Isoforms of PKC may have opposing effects on cytokine production. PD 98059, a compound that specifically inhibits the activation of mitogen‐activated protein kinase kinase (MEK1), inhibited TNF‐α production, but had insignificant effects on IL‐8 production. Pretreatment of PBMC with either PD 98059 or genistein reduced the extent of phosphorylation of p42 MAP kinase in cells activated on contact with IgG2. These findings suggest distinct signal transduction pathways for cytokine production in PBMC stimulated with IgG2. J. Leukoc. Biol. 64: 124–133; 1998.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141323/1/jlb0124.pd

Fragile site FS2 is a hotspot for initiation of BIR events resulting in LOH.

<p>The number of events initiated at each SNP is shown, and the two SNPs flanking fragile site FS2 are highlighted with a yellow box. The SNP indicated where each event maps is the first homozygous SNP in the stretch of homozygous SNPs. The BIR initiation site can be anywhere between the last heterozygous SNP and the first homozygous SNP. (A) The two homologs of the right arm of chromosome III are shown. The gray homolog is MS71-derived and contains fragile site FS2 and the <i>SUP4-o</i> allele. The red chromosome represents the YJM789-derived homolog. Large ovals represent the centromere. Black arrows on the chromosome diagrams indicate Ty1 elements. SNP markers used to map events are shown by circles and triangles on the chromosome diagrams. Triangles indicate a restriction site exists, circles indicate lack of the site. Numbers are the approximate chromosome coordinate in kb. The 66 BIR event initiation sites collected in Experimental Diploid #1 under low galactose conditions that cause replication stress are shown in the graph above the chromosome diagram. All BIR events in this diploid had three copies of the YJM789-derived SNPs, implying that the initiating lesion occurred on the gray, FS2-containing chromosome. (B) The 14 BIR event initiation sites collected in Experimental Diploid #1 under high galactose conditions that do not cause replication stress. (C) The 28 BIR event initiation sites collected in Experimental Diploid # 2 under low galactose conditions that cause replication stress. In this diploid, <i>SUP4-o</i> is located on the red, YJM789-derived chromosome. All BIR events in this diploid had three copies of the MS71-derived SNPs, implying that the initiating lesion occurred on the red, non-FS2-containing chromosome.</p

Instability at fragile site FS2 stimulates LOH by mitotic recombination and chromosome loss.

a<p>All diploids result from mating a MS71-derived haploid with a YJM789-derived haploid. Each haploid is isogenic with its parent strain, except for changes introduced by transformation (described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen.1003817.s003" target="_blank">Table S1</a>). MS71 is a <i>LEU2</i> derivative of AMY125 (<i>MATα ade5-1 leu2-3 trp1-289 ura3-52 his7-2</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen.1003817-Kokoska1" target="_blank">[64]</a>. YJM789 is a derivative of a clinical yeast isolate (<i>MAT</i>a <i>ura3 gal2 ho::hisG</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen.1003817-Wei1" target="_blank">[42]</a>. All diploids except AMC324 and AMC331 are homozygous <i>GAL-POL1</i>.</p>b<p>The frequency of reciprocal crossovers was calculated as 2*(number of crossover events/total colonies).</p>c<p>Only BIR events that result from a break on the chromosome containing the <i>SUP4-o</i> allele can be detected as a red/light pink sectored colony. Therefore, in Y332, Y382, AMC324, and AMC331, we report BIR resulting from breaks on the MS71-derived chromosome III. In AMC310, we report BIR resulting from breaks on the YJM789-derived chromosome III.</p>d<p>Only loss of the chromosome containing the <i>SUP4-o</i> allele can be detected as a red/light pink sectored colony. Therefore, in Y332, Y382, AMC324, and AMC331, we report loss of the MS71-derived chromosome III. In AMC310, we report loss of the YJM789-derived chromosome III.</p>e<p>The number in parenthesis is the 95% confidence interval. The number in brackets is the fold change from Y332 in high galactose. No local gene conversion at the <i>SUP4-o</i> locus was observed in any strain; therefore this category is not included in the table.</p

Locations of crossovers and BIR events in Control Diploids.

<p>For each diploid, the two homologs of the right arm of chromosome III are shown. The gray homolog is MS71-derived and the red homolog is YJM789-derived. Large ovals represent the centromere. Black arrows on the chromosome diagrams indicate Ty1 elements. SNP markers used to map events are shown by circles and triangles on the chromosome diagrams. Triangles indicate a restriction site exists, circles indicate lack of the site. Numbers are the approximate chromosome coordinate in kb. Crossover and BIR events are shown below the chromosomes. Black X's indicate crossovers that did not have gene conversion associated at the SNPs tested. Thin horizontal lines indicate 3∶1 conversion tracts associated with crossovers, and dotted lines indicate a non-adjacent conversion tract. Line color shows which chromosome was copied in gene conversion. BIR events are shown by arrowheads. The flat vertical edge of the arrowhead indicates the site at which the BIR was initiated; all BIR events extended to the end of the chromosome. The SNP indicated where each BIR event maps is the first homozygous SNP in the stretch of homozygous SNPs. The BIR initiation site can be anywhere between the last heterozygous SNP and the first homozygous SNP. The red color of the arrowheads indicates that the YJM789-derived homolog was the template for copying, implying that the initiating lesion occurred on the MS71-derived homolog. (A) Five crossover and eight BIR events mapped in Control Diploid #1, grown in high galactose. Both fragile site FS2 and the <i>SUP4-o</i> allele are located on the MS71-derived homolog, and this diploid is homozygous for the <i>POL1</i> gene under its native promoter. (B) One crossover and nine BIR events mapped in Control Diploid #2, grown in high galactose. This diploid is homozygous for the <i>POL1</i> gene under its native promoter, and the <i>NAT</i> gene replaces both Ty1 elements of fragile site FS2. (C) Three crossover and four BIR events mapped in Control Diploid #3 under replication stress caused by low levels of polymerase alpha. Fragile site FS2 has been inactivated in this diploid by expansion of the space between the two Ty1 elements of the fragile site.</p

Use of SNPs to map the location of mitotic recombination events.

<p>Single nucleotide polymorphisms (SNPs) between the two homologs of chromosome III that alter restriction sites were used to evaluate the type of event responsible for sectoring and to map the location of each event. Experimental Diploid #1 is shown. The gray chromosome represents the MS71-derived homolog and the red chromosome represents the YJM789-derived homolog. Centromeres are represented by large ovals and SNP sites by small ovals. FS2 is indicated by yellow stars and <i>SUP4-o</i> is represented by a gray rectangle. This strain is homozygous for the ochre-suppressible <i>ade2-1</i> mutation. (A) A BIR event that is stimulated by a lesion at FS2 is shown. The YJM789-derived homolog is used as a template for repair. After chromosome segregation in mitosis, the light pink cell remains heterozygous at all SNPs, while the red cell is homozygous for the YJM789 form of all SNPs distal to the invasion site. (B) A reciprocal crossover that occurs to repair a lesion at FS2 in S phase or G2 is shown. The crossover location is indicated by a black X. Transfer of genetic information from the YJM789-derived homolog during repair resulting in 3∶1 gene conversion of one SNP is shown in the yellow box. After chromosome segregation in mitosis, the white cell is homozygous for the MS71 version of SNPs distal to the crossover, while the red cell is homozygous for the YJM789 form of SNPs distal to the crossover, and the SNP within the region of gene conversion is homozygous in the red cell but heterozygous in the white cell.</p

Lesions at fragile site FS2 during S-phase initiate mitotic crossovers resulting in LOH.

<p>(A) Number of events at each SNP in Experimental Diploids #1 and #2 under replication stress. We summed the number of conversion tracts (both 3∶1 and 4∶0 events) crossing each SNP in both experimental diploids, and crossovers un-associated with a gene conversion were added to the sum of the closest centromere-distal SNP. Numbers are the approximate chromosome coordinate in kb for each SNP. (B) Locations of 41 crossovers and associated gene conversions collected in Experimental Diploid #1 under replication stress. Chromosome diagrams for Experimental Diploid #1 are the same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen-1003817-g004" target="_blank">Figure 4</a>. Crossover events are shown below the chromosomes. Black X's indicate crossovers that did not have gene conversion associated at the SNPs tested. A number in parenthesis indicates how many crossover events were at the site, if more than one. Thin horizontal lines indicate 3∶1 conversion tracts and thick lines indicate 4∶0 tracts. Dotted lines indicate a non-adjacent conversion tract. Line color shows which chromosome was copied in gene conversion. These crossover events were collected in two ways; 29 crossover events were collected among the colonies in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen-1003817-t001" target="_blank">Table 1</a>, and 12 crossover events were collected among another set of 14,792 colonies. (C) Locations of the 15 crossovers and associated gene conversions collected in Experimental Diploid #2 under replication stress. In this diploid, SUP4-o is located on the red, YJM789-derived homolog of chromosome III. (D) Locations of 5 crossovers and associated gene conversions in Experimental Diploid #1 in high galactose, which permits abundant production of polymerase alpha. These crossover events were collected in two ways; four crossover events were collected among the 30,543 colonies in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen-1003817-t001" target="_blank">Table 1</a>, and one crossover event was collected among another set of 4,792 colonies.</p