Protection from experimental allergic encephalomyelitis conferred by a monoclonal antibody directed against a shared idiotype on rat T cell receptors specific for myelin basic protein.

Immunizing Lewis rats with guinea pig myelin basic protein (MBP) yielded an encephalitogen specific, Ia-restricted, rat-mouse T cell hybridoma 5.10, which was used to establish a clonotypic mAb (10.18) that binds to and precipitates the rat TCR. By two-dimensional gel electrophoresis, the rat TCR was shown to consist of two disulfide-linked peptide chains with mol wt of 48,000 and 39,000. 10.18 binds the majority of cells in MBP-specific T cell lines that are capable of transferring experimental allergic encephalomyelitis (EAE) to Lewis rat recipients, but does not bind to either a purified protein derivative of tuberculin-specific cell line or an OVA-specific line. Furthermore, soluble 10.18 can block antigen-specific stimulation of hybridoma 5.10 but cannot control hybridomas, while immobilized 10.18 stimulates 5.10, but cannot control the hybrids. Though 10.18+ cells are very rare in normal rats, increase of 10.18+ cells is observed in MBP-primed paralyzed rats. Finally, when 10.18 is injected into MBP-primed Lewis rats, EAE is abrogated. We have thus characterized EAE as a "mono-idiotypic" autoimmune disease.

Molecular cloning and characterization of a novel neutrophil chemotactic factor from a filarial parasite.

To compare the molecular structure of a parasite-derived neutrophil chemotactic factor (NCF) with host-derived NCFs or other NCFs, molecular cloning of cDNA encoding NCF derived from Dirofilaria immitis adult worm (DiNCF) was performed. A D. immitis cDNA library was screened with an antibody to DiNCF, and one DiNCF cDNA clone (pD-4) was isolated. A fusion protein of pD-4 and gene 10 protein showed significant neutrophil chemotactic activity whereas gene 10 protein itself showed marginal neutrophil chemotactic activity. The total nucleotide sequence analysis revealed that pD-4 was 994 bp long with a 432 bp open leading frame encoding a 143 residue protein. The NH2-terminal amino acid sequence of the natural DiNCF and the deduced amino acid sequence from the cDNA showed that the mature functional protein was comprised of 112 amino acids. Although the deduced amino acid sequence of this protein did not show overall homology to host-derived NCFs or other known proteins, it contained a similar sequence (Met-Phe-Lys) to the known chemotactic peptides. The possibility of the functional epitope of DiNCF is discussed.

Hepatic eosinophilopoiesis from multipotent hemopoietic stem cells in Toxocara canis-infected mice.

Extramedullary hemopoiesis, recognized as hemopoietic foci, increased in the livers of Toxocara canis-infected mice. At the peak of the response (day-13 after infection), the majority of hepatic hemopoietic foci were of the eosinophil lineage. Hepatic nonparenchymal cells prepared from T. canis-infected mice on day 13 contained large numbers of hemopoietic stem cells, more than half of which were cycling. When W/Wv mice, which are genetically deficient in multipotent hemopoietic stem cells, were infected with T. canis, hepatic hemopoietic foci were rare throughout the course of infection. This impaired response of W/Wv mice was restored by bone marrow grafting from normal +/+ littermates. These results indicate that, in response to the increased demand, eosinophils are generated in the liver by the differentiation from multipotent stem cells, not only from the committed precursors.

Identification of a neutrophil chemotactic factor from Tritrichomonas foetus as superoxide dismutase.

Antibodies to a neutrophil chemotactic factor from Tritrichomonas foetus were used to screen a T. foetus cDNA expression library in lambda gt11. All positive clones were identified as homologs of iron-containing superoxide dismutase (SOD). Native gel electrophoresis showed that the antibodies indeed recognized T. foetus antigens with SOD activity. Two SOD genes were found in T. foetus, and cloned and sequenced as parts of larger genomic segments of 3844 and 4089 base pairs. Transcription initiated between the first and second methionine codons of each genomic open reading frame, generating mRNAs with 5' untranslated regions of 11-15 bases, and encoding proteins of 195 amino acids. The two SOD coding sequences lacked obvious introns. They were 79% identical at both the nucleotide and amino acid levels. Both SOD genes were inserted into a eukaryotic expression vector and stably expressed in mammalian cells; both proteins were recognized by the antibodies, and both assumed a cytosolic, extranuclear distribution in these cells. Histidine-tagged forms of both T. foetus SODs were expressed in E. coli and after purification, found to have neutrophil chemotactic activity similar to the non-recombinant factor purified from T. foetus. Identification of this neutrophil chemotactic factor as SOD provides additional insight into the host-parasite interaction.

Kinetic study of eosinophil chemotactic factor production with reference to eosinophilia and granuloma formation in mice infected with Schistosoma japonicum.

Kinetic changes of eosinophil chemotactic factor (ECF) production from granulomas, splenic T-cells or mast cells were examined with reference to granuloma formation around newly deposited single eggs in Schistosoma japonicum-infected mice. The peri-ovular granulomas began to appear at around 5 weeks post-infection (p.i.). Their size reached a peak at 6 weeks and then decreased gradually. Up to 8 weeks p.i., eosinophils were the predominant cell type in the granulomas. ECF-release from isolated granulomas paralleled the size of granulomas. Circulating ECF-A, which was assumed to be derived from mast cells, was also detected 6 weeks afterwards in parallel with the level of specific IgE antibody level against egg antigens in the serum. The circulating ECF-A peaked at 8 weeks and decreased after 10 weeks. Spleen cells began to produce ECF specific to bone-marrow eosinophils began at 5 weeks p.i., reached a peak at 6 weeks and then decreased rapidly. On the other hand, the production of ECF specific to eosinophils obtained from the peritoneal cavity began at 6 weeks and decreased rapidly thereafter. These results suggest that various kinds of host-derived ECFs seem to contribute, in one way or an other, to the accumulation of eosinophils in and around granulomatous lesions. The possible role of these ECFs in eosinophil mobilization from the site of production to the inflamed site is discussed.

A comparative study of the kinetic changes of hemopoietic stem cells in mice infected with lethal and non-lethal malaria.

The kinetic changes of hemopoietic stem cells in bone marrow and spleen were compared between lethal Plasmodium berghei- and non-lethal P. yoelii 17x-infected mice. P. yoelii 17x-infected mice showed more severe splenomegaly than those infected with P. berghei. P. yoelii 17x-infected mice also showed a greater degree of sustained increase in number of multipotent hemopoietic stem cells (colony-forming units in spleen: CFU-S) and committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) than P. berghei-infected mice. Such an increase was predominantly seen in the spleen of P. yoelii 17x-infected mice. In P. berghei-infected mice, the number of CFU-S, CFU-GM and also CFU-E only transiently increased and then decreased to a subnormal level at the late stage of infection. The proportion of cycling CFU-S was higher in P. berghei-infected mice than in P. yoelii 17x-infected mice. The IL-3 producing activity per spleen was much higher in P. yoelii 17x-infected than in P. berghei-infected mice at any point in time during the infection. Thus, hemopoietic changes seen after malaria infection seem to be closely related to the pathogenicity of the malaria parasite.

Non-specific immune suppression by CD8+ T cells in Brugia pahangi-infected rats.

Non-specific suppression of the immune response was investigated in Brugia pahangi-infected Lewis rats. The proliferative response of peripheral blood lymphocytes or splenic non-adherent cells to mitogens was significantly reduced by B. pahangi infection. The degree of hyporesponsiveness of splenic non-adherent cells to mitogens was comparable between microfilaremic and non-microfilaremic animals. The suppressed proliferative response of splenic non-adherent cells was restored by blocking with anti-CD8 monoclonal antibody. After separation of T cells into CD4+ and CD8+ subpopulations, only CD8+ T cells from B. pahangi-infected rats suppressed the proliferative response of normal spleen cells to concanavalin A. CD8+ T cells from normal rats had no suppressive effect. On the other hand, the proliferative response of CD4+ T cells to concanavalin A was comparable between normal and infected rats. These results suggest that CD8+ T cells participate in the non-specific suppression of immune response in experimental filariasis.

Eosinophil chemotactic factor in schistosome eggs: a comparative study of eosinophil chemotactic factors in the eggs of Schistosoma japonicum and S. mansoni in vitro.

Significant chemotactic activity for eosinophils was detected in soluble egg antigen (SEA) preparations of both Schistosoma japonicum and S. mansoni in dose-dependent fashion. The activity of S. japonicum SEA was higher than that of S. mansoni SEA. Gel filtration on Sephadex G-150 showed that S. japonicum SEA was composed of two groups of eosinophil chemotactic factors (ECFs), one of high molecular weight (JEE-H) and the other of low molecular weight (JEE-L). S. mansoni SEA showed ECF composition similar to that of S. japonicum SEA. JEE-H was stable on heating (100 degrees C, 60 min) and resistant to pronase digestion, but was sensitive to periodate oxidation. JEE-L was also stable on heating and resistant to pronase and carboxypeptidase A digestions. These properties of the ECFs were also held in common with those of S. mansoni SEA. JEE-L was extractable by toluene, indicating a hydrophobic nature. These results suggest that schistosome eggs themselves contain ECFs, and that the composition of S. mansoni and S. japonicum SEA-derived ECFs is essentially the same. However, they differ from the other ECFs which have already been described in schistosome infections.

In vitro and in vivo induction of neutrophil and eosinophil chemotactic responses by Schistosoma japonicum cercaria.

High neutrophil and eosinophil chemotactic activities of soluble extract of Schistosoma japonicum cercariae (SjCe-ext) were detected in vitro by using blind-well chemotaxis chambers and Millipore filters in a dose-dependent manner. Low doses (total of 0.25 and 2.5 micrograms) of SjCe-ext induced significant neutrophilic infiltrations in normal and S. japonicum-infected (8 week) guinea pig tissues beginning at 2 hr after the intradermal injection. Eosinophilic responses were observed in the infected animals especially in early phase (2-8 hr post-injection of SjCe-ext). SjCe-ext elicited strong immediate skin reactions (at 30 min) in the infected guinea pigs. Possible roles of these chemotactic activities and hypersensitivity reaction in S. japonicum infection are discussed.

Parasite-specific IgE and IgG levels in the serum and pleural effusion of paragonimiasis westermani patients.

In seven patients with paragonimiasis westermani, parasite-specific IgE and IgG levels in sera and pleural effusion were determined by an enzyme-linked immunosorbent assay (ELISA). The sensitivity to adult excretory-secretory (E-S) antigen was compared with the sensitivity to whole worm extract antigen, and the former was more sensitive in both an IgE-ELISA and IgG-ELISA. Both parasite-specific IgE and IgG could be detected by ELISA at levels much higher than those in control subjects using E-S antigen. When specific IgE and IgG levels in sera and pleural effusion of individual patients were compared, the latter had higher values. The difference between levels of specific IgE in pleural effusion and serum did not correlate with that of specific IgG. These results indicate that specific IgE and IgG antibodies form locally, i.e., in the lung, and that pleural effusions from patients with paragonimiasis are more suitable than serum for immunodiagnosis.

Leukocyte accumulation in sparganosis: demonstration of eosinophil and neutrophil chemotactic factors from the plerocercoid of Spirometra erinacei in vivo and in vitro.

In order to determine whether the plerocercoid of Spirometra erinacei itself has eosinophil and neutrophil chemotactic factors, in vivo and in vitro examinations were carried out. We could observe large numbers of eosinophils and neutrophils which accumulated at the injection site of normal guinea pig skin following intradermal injection of soluble extract of plerocercoids of S. erinacei. At 1 hour after the injection, neutrophils appeared at the site, and the cell number reached its peak at 4 hours. Eosinophils appeared rather later than neutrophils (at 2-4 hours), and the number of cells reached its peak at 8 hours after the injection. Eosinophil and neutrophil chemotactic activities were also confirmed in an in vitro system by using a blind-well chemotaxis chamber with a Millipore filter in dose dependent fashion. An eosinophil chemotactic factor (ECF) with molecular weight of approximately 15,000, and two different neutrophil chemotactic factors, one of about same molecular weight as the ECF and the other of low molecular weight, were demonstrated by gel filtration on Sephadex G-200. Furthermore, it was confirmed that those factors were released from parasite by the detection of intensive eosinophil and neutrophil chemotactic activities in the culture supernatants containing plerocercoids.

Detection of high molecular weight eosinophil chemotactic factor in murine schistosomiasis sera.

Eosinophil chemotactic activity in sera from mice undergoing an acute stage of schistosomiasis japonica and mansoni was examined. Eosinophilotactic activity in the serum was dependent on the dose and time of infection. Eosinophilotactic activity in sera from S. japonicum-infected mice was higher than that from S. mansoni-infected mice when they were compared at the comparable dose and time of infection. After gel chromatography on Sephadex G-200, eosinophilotactic activity in sera from mice infected with 30 cercariae of S. japonicum for 5 weeks was detected in the high molecular weight component. On the other hand, when sera from mice infected with 30 cercariae of S. japonicum for 8 weeks was chromatographed through Sephadex G-200 columns, eosinophilotactic activity was segregated into high (greater than 455,000) and low (less than 13,000) molecular weight components. High molecular weight ECF in sera from mice infected with 30 cercariae of S. japonicum for 8 weeks had high affinity to Con A, and was stable to heating or pronase digestion, but was sensitive to periodate oxidation, indicating its polysaccharide or glycoprotein nature. This high molecular weight ECF could be adsorbed by, and eluted from immunoaffinity beads coated with rabbit IgG anti-S. japonicum adult worm antibody. Thus, at least some part of circulating high molecular weight ECF would be derived from adult parasites.

Purification and characterization of a neutrophil chemotactic factor from Dirofilaria immitis.

A neutrophil chemotactic factor (NCF-Di) was purified from a crude extract of Dirofilaria immitis adult worm by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. NCF-Di showed a single protein band by both polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. The molecular weight of NCF-Di was estimated to be 17,000 by gel filtration on Sephadex G-150, and 14,000 by SDS-PAGE. NCF-Di was an acidic protein with isoelectric point of 4.5. NCF-Di was absorbed neither to lentil lectin-Sepharose nor to concanavalin A-Sepharose. The chemotactic activity of NCF-Di was heat labile (56 C, 1 hr), but was resistant to periodate oxidation. These results suggest that NCF-Di is a simple peptide which has few or no sugar chains. These physicochemical properties of NCF-Di were compared to previously reported parasite-derived chemoattractants or purified allergen of D. immitis.

The localization of allergens of Paragonimus westermani by pleural exudates from patients.

The allergens of the lung fluke Paragonimus westermani were localized by indirect immunostaining in adult fluke sections using pleural exudates from 3 patients with P. westermani. Immunostaining performed by using pleural exudate with the highest level of specific IgE revealed that the P. westermani major allergen (or allergens) was located in the gut epithelium and luminal contents and that minor allergens were in the tegument and parenchyma. The antigens recognized by specific IgG were located at various sites including those recognized by specific IgE. Paragonimus westermani-specific IgE cross-reacted with only the gut of 2 other Paragonimus species, Paragonimus miyazakii and Paragonimus ohirai. The major allergen in the gut also was recognized by the other 2 pleural exudates. These results indicate that the substance present in and secreted from the gut is not only a major allergen but is also a common allergen among Paragonimus species.

Eosinophil and neutrophil chemotactic activities of adult worm extracts of Schistosoma japonicum in vivo and in vitro.

Large numbers of eosinophils and neutrophils attracted to the soluble extract of Schistosoma japonicum adult worms (SjAW-ext) were detected at the injection site of normal guinea pig skin. Eosinophil and neutrophil chemotactic activities were also confirmed in in vitro assay by using blind-well chambers with Millipore filters in dose-dependent fashion. Two components of SjAW-ext showed eosinophil chemotactic activity; one was in the high molecular weight fraction (JAE-H), estimated to be more than 440,000 daltons, the other in the low molecular weight fraction (JAE-L) obtained by Sephadex G-200 gel filtration. High neutrophil chemotactic activity was detected in the JAE-L. These eosinophil and neutrophil chemotactic activities were also detected in culture fluid of S. japonicum adult worms. Eosinophil chemotactic factor (ECF) of JAE-H was stable to heating (100 C, 30 min) and pronase digestion, but completely destroyed by periodate oxidation. It is suggested that the ECF of JAE-H is a glycoprotein. JAE-L was also stable to heating (56 and 100 C, 30 min) and pronase digestion for eosinophil chemotaxis. Possible roles of those activities in schistosome infections are discussed.

Bone marrow and spleen cell colony formation in mice infected with Schistosoma japonicum.

Progenitor cells in the bone marrow and the spleen of mice, whether infected with Schistosoma japonicum or not, formed cell clusters and colonies when incubated with culture supernatant fluid of spleen cells incubated with soluble egg antigen (SEA). The egg extract, up to a concentration of 250 micrograms/ml protein, did not directly stimulate progenitor cell proliferation in the bone marrow. Eosinophilia in mice infected with S. japonicum may be mediated indirectly by egg antigen-stimulated immune lymphocytes and not directly by the egg antigen.

Neutrophilic nodules in the intestinal walls of Japanese monkeys associated with the neutrophil chemotactic activity of larval extracts and secretions of Oesophagostomum aculeatum.

High neutrophil chemotactic activity was detected in the culture medium from Oesophagostomum aculeatum larvae in vitro using blind-well chambers with Millipore filters, and guinea pig leucocytes as indicator cells. Neutrophil chemotactic activity was also detected in the extract from larval worms in a dose dependent fashion. This activity was detected in the low molecular weight fractions adjacent to a sodium chloride marker by gel filtration on Sephadex G200. These results were further confirmed with monkey neutrophils. The possible role of this activity in the formation of granulomatous lesions rich in neutrophils found in O aculeatum infections in the Japanese monkey is discussed.

Partial purification and characterization of eosinophil chemotactic factors from soluble extract of Fasciola species.

Eosinophil chemotactic factor (ECF) was partially purified from common liver flukes (Japanese strain of Fasciola sp) by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. The molecular weight of ECF was estimated to be approximately 27,000 by high-pressure liquid chromatography. The ECF was heat labile (56 and 100 C for 30 minutes) and was not bound with lentil-lectin Sepharose. The results of isoelectric focusing showed that ECF comprises at least 2 components; one was a major ECF with an isoelectric point of 3.1, and the other, a minor ECF with an isoelectric point of 3.8 to 4.5. These results indicate that ECF of common liver flukes are acidic proteins.

Temporary appearance of a circulating granulocyte-macrophage colony-stimulating factor in lethal murine malaria.

Infection of mice with Plasmodium berghei engendered a temporary appearance of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the serum. The peak of GM-CSF levels was detected at day 2 post-infection, and then gradually decreased. On the other hand, the number of committed stem cells for granulocytes and macrophages (CFU-GM) in bone marrow transiently decreased at day 2 post-infection, and then increased and peaked at day 6 post-infection. When the serum of P. berghei-infected mice was fractionated by gel chromatography on Sephacryl S-300, GM-CSF activity was detected as a single peak with an apparent molecular weight of 64 KDa. GM-CSF was entirely adsorbed to concanavalin A-Sepharose 4B affinity chromatography, and was sensitive to pronase digestion, indicating its glycoprotein nature. These results suggest that the circulating GM-CSF would contribute the increase of granulocyte-macrophage hemopoiesis in the early phase of malaria.

Depressed specific and nonspecific immune responses in secondary Brugia pahangi infection in jirds.

The immune responsiveness to specific antigens or mitogens was examined in jirds after primary and secondary infections with Brugia pahangi. When spleen cells were obtained from secondarily infected jirds, their proliferative responses to mitogens such as Con A or LPS, or to specific antigens prepared from infective larvae or adult worms were significantly lower than those of spleen cells obtained from primarily infected jirds. The proliferative responses of the peripheral blood mononuclear cells obtained from animals undergoing primary and secondary infections also showed a similar tendency. The depressed proliferative responses of the secondary infected spleen cells to Con A or LPS was partially restored by removing adherent/phagocytic cells from the original cell populations. After deletion of the adherent cells, however, antigen-specific proliferative responses were not altered and remained at low level. These results suggest that at least two different mechanisms of depression, namely adherent cell-mediated antigen-nonspecific suppression and unresponsiveness of lymphocytes to filarial antigens, are induced in jirds in the secondary infection.