Yoon Berm Kim

Intravenous Immunoglobulin Inhibits Natural Killer Cell Activity In Vivo in Women With Recurrent Spontaneous Abortion

We previously reported elevation of natural killer (NK) cells in women with recurrent spontaneous abortion (RSA) of immune etiology. In this study, we investigated the effect of intravenous immunoglobulin G (IVIg) on peripheral blood NK activity in vivo in women with RSA. Blood was drawn prior to and 7–11 days after IVIg therapy in eight women with RSA. NK activity was measured using K562 as target cells for 51Cr‐release assays. Serum IgG concentrations were also measured. All received 400 mg/kg/day of IVIg for 3 consecutive days. 1) Seven of eight women became pregnant. Five delivered a live born infant. Three out of five women (60%) who delivered a live born infant showed a significant inhibition of NK cytotoxicity post IVIg and the rest did not show any changes; 2) NK cytotoxicity was significantly increased in a woman who miscarried again; 3) A woman who miscarried a chromosomally abnormal fetus showed a significant inhibition of NK cytotoxicity after IVIg; and 4) Serum IgG concentration increased significantly from 9.3 ± 3.0 mg/ml to 23.5 ± 5.1 mg/ml post IVIg therapy. IVIg effectively inhibits peripheral blood NK activity in vivo. These results are consistent with our previous finding showing that IVIg inhibits NK cell activity in vitro. Women with RSA and elevated NK cells may benefit from IVIg treatment.

Phenotype of porcine monocytic cells: modulation of surface molecule expression upon monocyte differentiation into macrophages

When porcine blood monocytes differentiate in vitro into macrophages, their morphology, as well as side scatter and forward scatter measured by flow cytometry, changed in a manner similar to that with human cells. During this differentiation, the initial high expression of CD molecules on porcine monocytes was down-regulated, with one exception--SWC9. Freshly isolated blood monocytes were SWC9-, but after culture the cells had become SWC9+. Thus, porcine monocytes were characterised as SWC3+SWC9-CD14high; macrophages were SWC3+SWC9+CD14low, the latter also displaying a down-regulation of CD11a/18, and, to a lesser degree, CD44. Both SWC9-CD14high monocytes and SWC9+CD14low macrophages were identifiable in freshly prepared monocytic cells from the spleen. Alveolar macrophages, on the other hand, were dominated by SWC9+CD14low cells, similar in phenotype to in vitro monocyte-derived macrophages. The consequences which these results have for studies on virus infections of porcine monocytic cells are discussed.

Workshop studies on monoclonal antibodies reactive against porcine myeloid cells

Investigators from eight laboratories analyzed the reactivity of 22 monoclonal antibodies (mAb) against porcine myeloid cells. Based on binding data, clustering analysis and inhibition studies, workshop mAb 74-22-15 (003) and 6F3 (007) were assigned a swine workshop cluster number 3 (SWC3). These mAb recognized macrophages and neutrophils; neutrophils; a monocyte/macrophage-specific mAb was not identified by this workshop.

Analyses of monoclonal antibodies reactive with porcine CD44 and CD45

Twenty-six monoclonal antibodies (mAbs), assigned to the CD44/CD45 section of the First International Swine CD Workshop, were compared for their reactivity against a selected group of target cells by one- and two-color flow cytometric analysis. Based on staining and reactivity patterns the 26 mAbs were assigned to six groups, group F1 mAbs were designated CD44 mAbs; and groups F2 and F3 as CD45 mAbs. With the information available, a CD designation could not be given to the mAbs in groups F4, F5 or F6 consisting of four, three and four mAbs each, respectively. The reactivity of all six mAbs in group F1 (MAC35, 25-32, PORC24A, H22A, BAG40A, and BAT31A) was blocked by soluble porcine CD44. One mAb in this group (MAC325) reacted with a cell surface protein with a molecular weight of 80 kDa and was designated as CD44; the other five mAbs were designated as wCD44 because no molecular weight was known. Blocking experiments utilizing a cross reactive anti-human CD44 (mAb Z062) allowed the definition of the wCD44a epitope recognized by mAbs PORC24A and H22A. The group F2 mAbs (74-9-3; MAC323; K252.1E4; and 2A5) were designated as CD45 based on their broad reactivity pattern with lymphoid and myeloid cells and their ability to immunoprecipitate three polypeptides with an apparent molecular weight of 226, 210 and 190 kDa. The F3 mAbs (MAC327; MAC326; 3a56 and -a2) were designated as CD45R based on their restricted reactivity against lymphoid and myeloid target cells, and their ability to immunoprecipitate either two polypeptides with an apparent molecular weight of 226 and 210 kDa (mAbs MAC327 and MAC326) or a single polypeptide with an apparent molecular weight of 210 kDa (mAbs-a2 and 3a56). Sequential immunoprecipitation analyses confirmed the relatedness of the F2 and F3 group mAbs. The work conducted for this first workshop led to the definition of six mAbs specific for CD44, four mAbs specific for CD45, and four mAbs specific for CD45R which should prove to be very valuable reagents for the study of the porcine immune system.

Sequence analysis of porcine immunoglobulin light chain cDNAs

A porcine cDNA library was constructed using poly(A)+ RNA isolated from the spleen of an adult Minnesota miniature swine. Screening the library with antisera specific for porcine immunoglobulin light chains resulted in the selection and isolation of two recombinant clones, PLC18 and PLC3, which encode for kappa and lambda light chains, respectively. These cDNAs contain sequence information for a portion of the variable region and all of the constant region. The lengths of the constant regions are 105 amino acids for lambda and 108 amino acids for kappa. The deduced amino acid sequences of porcine immunoglobulin light chains share a high degree of homology with similar sequences from other species in both the fourth framework region and the constant region.

Porcine myelomonocytic markers: summary of the second international swine CD workshop

Forty five mAbs submitted to the Second International Swine CD workshop were analyzed by six different laboratories for their possible reactivity with porcine myelomonocytic cells using flow cytometry and immunohistochemistry. As a result of these analyses, a new swine workshop cluster, SWC9, composed of two mAbs that recognize an antigen selectively expressed on mature macrophages, was defined. In addition, several mAbs were identified, allowing the differentiation of granulocytes from monocytes/macrophages, or monocytes from macrophages. Further work is required to identify the antigen recognized by these mAbs. Nevertheless, they should already prove useful for the identification of different stages in the macrophage maturation/differentiation, and will certainly aid analyses on the complexity of the mononuclear phagocyte system in the pig. Finally, the cross-reactivity of three anti-human CD14 mAbs with porcine myelomonocytic cells was established in this workshop.

Establishment and characterization of endothelial cell lines from the aorta of miniature pig for the study of xenotransplantation

Pig endothelial cells are the first cells to interact with human immune components after organ xenotransplantation, which is a procedure currently considered to be the best treatment option for end‐stage organ failure. It is, therefore, essential to study the mechanisms of molecular interaction between pig endothelial cells and human immune components, in order to overcome xenograft rejection. The aim of this study was to establish immortalized pig aortic endothelial cell lines, in order to facilitate future in vitro studies of human anti‐pig immune responses. Endothelial cell lines were established following the transfection of primary endothelial cells isolated from the aortas of the Minnesota miniature pig with plasmid pRNS‐1 carrying genes for neomycin resistance and the SV40 large T antigen. The immortalized cell lines showed a relatively rapid doubling time (17.6 h) and the endothelial cell phenotype, as indicated by the formation of typical cobblestone monolayers and by the constitutive expression of PECAM‐1 and the von Willebrand factor. Flow cytometric analysis demonstrated the constitutive expression of SLA class I and CD86, whereas the expression of E‐selectin and SLA class II was only induced after stimulation with human TNF‐α and pig IFN‐γ, respectively. On the other hand, no CD80 expression was detected in the primary cells or cell lines in the presence or absence of either human TNF‐α or pig IFN‐γ. A vigorous human T cell proliferation against these cell lines was observed in the mixed lymphocyte—endothelial cell culture. These results suggest that pig endothelial cells, immortalized by the introduction of SV40 T, retain their original characteristics, except for the acquired property of immortalization, and that they may be useful for future in vitro studies of xenogeneic human anti‐pig immune responses.

Workshop studies with monoclonal antibodies identifying a novel porcine differentiation antigen, SWC9

Two monoclonal antibodies (mAb) within cluster M4 of the myeloid section of the Second International Swine CD Workshop, C4 (No. 144) and PM18-7 (No. 192), showed reactivity with thymocytes and among cells of myelomonocytic origin with mature macrophages but not with monocytes and granulocytes. Both mAb recognize a protein showing two bands of 205 kDa and 130 kDa under both reducing and non-reducing conditions. Although epitope mapping with these mAb could not be performed, this cluster received the SWC9 designation.

Two distinct cytolytic mechanisms of macrophages and monocytes activated by phorbol myristate acetate.

Pulmonary alveolar macrophages (PAM) and peripheral blood monocytes (PBMO) of the miniature swine can be converted to cytolytically active effector cells by treating with phorbol myristate acetate (PMA) as determined by enhancement of cytotoxicity to various target cells. Kinetics of the PMA‐activated PAM and PBMO in cytotoxicity show that the effective PMA concentration ranges from 10 to 1,000 ng/ml. Induction of cytotoxic macrophages and monocytes occurred as early as 30 min and to their maximum cytotoxicity after 1 hr exposure to PMA and the enhanced cytotoxic activity persisted up to 24 to 40 hr when PMA was removed by washing after 1 hr exposure, but prolonged exposure to PMA for more than 6 hr resulted in a drastic decrease of cytolytic activities suggesting the prolonged exposure to PMA causes macrophages and monocytes to become refractory to PMA stimulation. Target cells displayed varying degrees of cytotoxic sensitivity to the PMA‐activated PAM and PBMO; PRBC, SRBC, and K562 were sensitive, WEHI‐164 and U937 were relatively sensitive, and SB was very resistant to these activated effector cells. The mechanisms of PMA‐induced cytotoxicity could largely be divided into two categories. One was the H2O2 mediated killing as shown by complete reduction of cytotoxicity after adding catatase in the assay. The other was the proteases mediated cytolysis, which could be blocked by protease inhibitors, Phenyl methyl sulfonyl fluoride (PMSF), and N‐ α‐p‐tosyl‐L‐lysine chloromethyl ketone (TLCK). H2O2 was the only mediator produced in large enough quantities from PBMO to kill target cells, whereas PAM could produce both mediators (H2O2 and proteases). PRBC, SRBC, and K562 appeared to be killed by H2O2 produced by PAM and PBMO. In contrast, U937 and WEHI‐164 appeared to be killed by proteases in PAM mediated cytolysis but by H2O2 in PBMO‐mediated cytolysis. These results suggest that the observed cytolytic mechanisms can be differed by type of target cells as well as the source of mononuclear phagocytes within the individual animal.

Competitive binding analysis of monoclonal antibodies reactive with porcine alveolar macrophages using anti-CD14 and anti-CD18.

Four monoclonal antibodies (mAb) from the myeloid subset panel of the First International Swine CD Workshop (74-22-15, DH59B, PM16-6, and MUC21A) were analyzed using competitive inhibition studies with anti-human CD14 (My4) and anti-human/anti-porcine CD18 (MHM23) on porcine alveolar macrophages. Results suggested that none of the mAb tested recognized CD14 or CD18 on porcine alveolar macrophages. Additionally, the cross-reactivity of My4 with porcine alveolar macrophages was established.