Shuichi Furusawa

Immunoglobulin gene hyperconversion ongoing in chicken splenic germinal centers.

It has been believed that the peripheral lymphocytes in chickens proliferate by self‐renewing amplification of the preimmune repertoire generated in bursa. We amplified rearranged immunoglobulin variable (V) region genes from the single germinal centers induced by immunization. The sequence analysis of these genes revealed that most were derived from distinct B‐cell clones which expanded locally, generating somatic antibody mutants at a high rate. Somatic hypermutations included unlinked base changes and the linked base modifications interpreted as unidirectional transfer of sequences from V region pseudogenes. This finding demonstrates the ongoing post‐bursal diversification of B‐cells in splenic germinal centers by templated gene conversion as well as untemplated point mutations.

Studies on Murine IgE with Monoclonal Antibodies

Rat monoclonal antibodies were constructed by fusion of immunized rat spleen cells with a nonsecreting mouse myeloma cell. Two monoclonal antibodies (6HD5 and HMK-12) were selected for further study. Both reacted with various IgE molecules of different specificities and different allotypes, but did not react with immunoglobulins of other isotypes and with light chains. These antibodies were therefore anti-isotypic (IgE) and not anti-allotypic or anti-idiotypic. It was shown by competition studies that these antibodies recognize different epitopes on the FcR epsilon fragment. A sensitive ELISA for the quantitation of murine IgE was developed with these monoclonal antibodies; the sensitivity was between 2 and 250 ng/ml for detection of serum IgE levels. Good correlation was obtained with protein amounts as determined by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA) activities. Both monoclonal antibodies were used to study anaphylactic reactions elicited by IgE antibodies. Both could inhibit PCA reactions and both could elicit reverse PCA reactions.

Role of bursin in the development of B lymphocytes in chicken embryonic Bursa of Fabricius

Localization and role of bursin during Bursa of Fabricius (BF) ontogeny were examined by immunohistochemical staining and by in ovo injection with anti-bursin antibody. Mouse monoclonal anti-bursin antibody HU2 was generated by immunization with synthetic bursin. It recognized reticular cells (REC), follicular associated epithelium (FAE), FAE-supporting cells, and the basal layer of interfollicular epithelium (IFE) in the mature BF. Bu-1(+) cells were first detectable in the mesenchyme area at 13 days of embryogenesis (E13) before bud formation, then lined up along the bud, and homed into the bud at around E15. IgM(+) cells were detected in the bud after E13. Bursin was first observed at the under edge of the bud. Injection of HU2 into embryonal vein at E13 suppressed the appearance of IgM(+) cells in the Bursa at E17. These results indicate that bursin exists beneath the bud and may act on the appearance of IgM(+) cells during BF ontogeny.

Suppressive effect of intravenous immunoglobulins on the activity of interleukin-1

In order to study the effect of human immunoglobulin preparations for intravenous use (IVIg) on the production and activity of interleukin-1 (IL-1) derived from monocytes, we treated cultured monocytes with IVIg and examined the lymphocyteactivating factor (LAF) activity of IL-1 in the culture supernatants. The results showed that IVIg suppressed the activity from most healthy adults and some febrile children with acute respiratory disease or Kawasaki disease. Further studies revealed that intact Ig (whole molecular Ig) did not suppress the mRNA expression of IL-1α or IL-1β in mononuclear cells, that intact Ig and pepsin-digested Ig inhibited the LAF activity of recombinant IL-1 (rIL-1) and also that intact Ig contains immunoglobulin (probably anti-IL-1 antibody) which binds with rIL-1 by dot blotting using biotin-streptavidin. These results suggest that IVIg suppresses neither IL-1 synthesis nor the release of IL-1 from monocytes but does neutralize IL-1α and IL-1β activity by binding IL-1 proteins as an anti-IL-1 antibody.

Construction of recombinant monoclonal antibodies from a chicken hybridoma line secreting specific antibody

The chicken is a useful animal for the development of the specificantibodies against the mammalian conserved proteins. We generated twotypes of recombinant chicken monoclonal antibodies (mAbs), using a phagedisplay technique from a chicken hybridoma HUC2-13 which secreted themAb to the N-terminal of the mammalian prion protein (PrP). Althoughthe mAb HUC2-13 is a useful antibody for the prion research, thehybridoma produces a low level of antibody production. In order to producea large amount of the mAb, we have constructed a single chain fragmentvariable region (scFV) mAb by using the variable heavy(VH) and light (VL)genes which were amplified by using the two primer pairs and theflexible linker. The two phage display mAbs (HUC2p3 and HUC2p5)expressed on a M13 filamentous phage and their soluble type mAbs(HUC2s3 and HUC2s5) were reacted with the PrP peptide antigen in theELISA. In the Western blot analysis, the mAbs HUC2p3 and HUC2s3 wereas reactive to PrPc from mouse brains as the mAb HUC2-13 was. The nucleotide sequences of VH and VL genes from HUC2-13 and the two cloneswere identical except for only one residue. These results indicate that themethods presented here provide an effective tool for the improvement ofthe low levels of antibody production in the chicken hybridoma system.

Immunobiology of chicken germinal center: I. Changes in surface Ig class expression in the chicken splenic germinal center after antigenic stimulation

The germinal center (GC) develops after antigenic stimulation and is thought to occur at the site of various immune responses. We separated a single GC from chicken spleen after antigenic stimulation. Flow cytometric analysis of the cells derived from a single GC and RT-PCR analysis of Ig mRNA expression in GC was performed. Direct evidence indicates that: (1) there was a considerable difference in the cell population of each GC, (2) the ratio of CD3(+) cells in a GC remains constant at 10-20%, (3) the highest proportion of sIgY(+) cells in a GC occurs 1 week after the time of highest proportion of sIgM(+) cells, and (4) RT-PCR analysis was used to detect IgY mRNA expression in a GC. The continuous existence of CD3(+) cells, the alterations in sIgM(+) and sIgY(+) cell ratios, and the expression of IgY mRNA strongly suggest that Ig class switching occurs in the GC during an immune response.

Development of maternal IgG-free chick obtained from surgically bursectomized hen

IgG-free eggs and chicks were developed, so as to study the role of maternal IgG in the development of the immune system. Surgical bursectomy on the 18th day of incubation deprived chickens of B cells and eliminated IgG synthesis. Bursectomized chickens are usually dead before sexual maturity under conventional conditions. When surgically bursectomized chickens were housed in an isolated clean room and antibiotics were administered to them, they could survive to sexual maturity. Finally, we succeeded in obtaining IgG-free fertilized eggs and maternal IgG-free chicks from surgically bursectomized hens. The amount of yolk IgG in IgG-free eggs was one-ten thousandth less than that in normal eggs. The level of IgM in the serum of maternal IgG-free chicks reached six times higher than that of normal chicks 5 days after hatching.

Molecular cloning and expression of canine interleukin 8 cDNA

Molecular cloning of canine interleukin-8 (IL-8) was performed to establish a basis for its investigation in the canine immune system. From a cDNA pool constructed from LPS-stimulated popliteal lymph node cells, canine IL-8 cDNA covering the whole coding region was amplified by polymerase chain reaction. The nucleotide sequence of a canine IL-8 clone, designated pcIL-8#38, was highly similar to those of human, rabbit and porcine IL-8, and comprised 353 bp with an open reading frame that encoded 101 amino acids. Analysis of the deduced amino acid sequence of insert DNA in pcIL-8#38 showed 76.5, 80.2, and 87.0% similarities with human, rabbit and porcine IL-8 proteins, respectively. Insert DNA of pcIL-8#38 was transferred to a mammalian expression vector, pcDL-SR alpha 296, and transfected into Cos7 cells. The supernatant of the transfectant had neutrophil chemotactic activity when it was examined by the neutrophil migration assay, suggesting that our cloned cDNA was biologically active. The cloned canine IL-8 cDNA will be useful for canine inflammatory disease and comparative immunology research.

Production of interleukin-1-alpha and -beta by human peripheral polymorphonuclear neutrophils.

The mechanism of the production of interleukin-1 (IL-1) by human peripheral polymorphonuclear neutrophils (PMN) was investigated. Supernatants of PMN stimulated with 30 micrograms/ml lipopolysaccharide (LPS) were used as extracellular IL-1 and supernatants of their lysate as intracellular IL-1 source. IL-1 activity was measured by the C3H/HeJ thymocyte co-mitogenic assay. The supernatants from PMN stimulated with LPS for 72 h showed IL-1 activity which had an apparent molecular weight of 15-20 kilodaltons and pI of 5.0 and more than 8.5. It was neutralized with anti-IL-1 antibodies and it lacked IL-2 activity. Our time course study of the IL-1 assay with neutralization by anti-IL-1-alpha and -beta antibodies indicated that the extracellular IL-1-beta activity appeared predominantly in the early incubation periods, whereas alpha activity appeared predominantly in the late periods. Intracellular IL-1-alpha but not beta activity was detected mainly at the intermediate incubation periods. These data indicate that PMN stimulated with LPS produce both IL-1-alpha and -beta, and release IL-1-beta first and IL-1-alpha later.

(免疫学)ニワトリ胚におけるバイエル氏板,盲腸扁桃(腸管付属リンパ組織)の発生

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyers patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckels diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.