bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of primary Strongyloides ratti infections in mice: a role for interleukin‐5
Authors: OVINGTON  , MCKIE  , MATTHAEI  , YOUNG  , BEHM
Published: March 2003

C57BL/6 mice genetically deficient in interleukin‐5 (IL‐5−/−) and normal C57BL/6 (IL‐5+/+) mice were infected with larvae of a homogonic strain of the nematode Strongyloides ratti. In primary infections both male and female IL‐5−/− mice released two to four times more eggs and larvae than IL‐5+/+ mice. IL‐5−/− mice harboured about 60% more intestinal worms, which were more fecund, than IL‐5+/+ mice. The duration of the infection was similar in normal and IL‐5‐deficient mice. Both IL‐5−/− and IL‐5+/+ mice resisted a secondary infection. IL5−/− mice lost more weight during the infection than normal mice and took longer to regain their initial weight after expelling the worms. The number of eosinophils increased in the bone marrow, peritoneal cavity and small intestine of IL‐5+/+ mice, but not IL‐5−/− mice, following infection. No significant differences between infected IL‐5+/+ and IL‐5−/− mice in mast cells or other leucocytes were observed in the peritoneal cavity. Thus, IL‐5 functions to protect the host in a primary infection of S. ratti by limiting the number and fecundity of worms establishing in the small intestine. This protection is correlated with elevated blood and tissue eosinophilia which occurs in normal mice but not in IL‐5−/− mice.
Distinct functions of interferon‐γ for chemokine expression in models of acute lung inflammation
Authors: NEUMANN  , EMMANUILIDIS  , STADLER  , HOLZMANN
Published: March 2003

Challenge of the immune system with bacterial superantigens or endotoxin induces the systemic release of cytokines followed by lethal septic shock. The lung is particularly susceptible to systemic toxin exposure resulting in acute leucocyte infiltration and vascular damage. In the present study, the functions of interferon‐γ (IFN‐γ) and tumour necrosis factor (TNF) for chemokine regulation during acute lung inflammation were examined. Following administration of the superantigen, staphylococcal enterotoxin B (SEB), lung mRNA levels of the chemokines cytokine‐induced neutrophil chemo‐attractant (KC), lipopolysaccharide‐induced CXC chemokine (LIX), macrophage chemotactic protein‐1 (MCP‐1), macrophage inflammatory protein (MIP)‐1α and MIP‐2 were increased to a similar extent both in controls and in mice deficient for the IFN‐γ or 55 000 MW TNF receptors. In contrast, interferon‐inducible protein‐10 (IP‐10) and monokine induced by IFN‐γ (Mig) mRNA expression was markedly reduced in mice deficient for IFN‐γ or IFN‐γ receptor, but not in 55 000 MW TNF receptor knockout mice. In situ hybridization experiments demonstrated that IP‐10 was highly expressed in lung interstitial macrophages of C57BL/6, but not of IFN‐γ receptor‐deficient mice. In contrast to SEB administration, treatment with lipopolysaccharide resulted in a strong induction of IP‐10 and Mig in IFN‐γ receptor‐deficient mice. Together, these results establish a critical function of IFN‐γ for chemokine induction in acute lung inflammation that is dependent on the nature of the inflammatory stimulus.
Role of inducible nitric oxide synthase in the regulation of neutrophil migration in zymosan‐induced inflammation
Authors: AJUEBOR  , VIRÁG  , FLOWER  , PERRETTI  , SZABÓ
Published: March 2003

In the present study, by comparing the responses in wild‐type mice and mice lacking the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the role played by iNOS in the regulation of polymorphonuclear granulocyte (PMN) accumulation and chemokine production in the mouse peritoneal cavity in response to administration of zymosan (0·2 mg). Zymosan injection induced the production of nitric oxide, and triggered a time‐dependent PMN immigration into the peritoneal cavity. This response was associated with increases in the level of the chemokines macrophage inflammatory protein (MIP)‐1α, MIP‐2, monocyte chemo‐attractant protein (MCP)‐1 and cytokine‐induced neutrophil chemo‐attractant (KC), as measured in the peritoneal cavities. Injection of zymosan also induced a time‐dependent increase in the production of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) in the peritoneal cavity. When comparing the response between wild‐type and iNOS knockout (KO) mice, we observed that the low‐level PMN accumulation measured at 1 hr was slightly but significantly increased in the absence of functional iNOS. On the other hand, the delayed response (2–4 hr after zymosan) of PMN accumulation was suppressed in the iNOS KO mice. The early enhancement of PMN infiltration in the iNOS‐deficient mice was associated with increased peritoneal levels of MIP‐2, KC and IL‐10 proteins. The delayed suppression of PMN infiltration was associated with reduced MIP‐2 and IL‐10 levels in the peritoneal cavity. The lack of iNOS did not affect the release of MIP‐1α and MCP‐1 at any of the time‐points studied. The current data demonstrate that iNOS regulates the production of certain CXC (but not CC) proinflammatory chemokines, the production of IL‐10 and exerts a biphasic regulatory effect on PMN accumulation in zymosan‐induced acute inflammation.
Reciprocal effects of interleukin‐4 and interferon‐γ on immunoglobulin E‐mediated mast cell degranulation: a role for nitric oxide but not peroxynitrite or cyclic guanosine monophosphate
Authors: DESCHOOLMEESTER  , EASTMOND  , DEARMAN  , KIMBER  , BASKETTER  , COLEMAN
Published: March 2003

We report that cultured rat peritoneal cells spontaneously synthesize nitric oxide and this is associated with active suppression of mast cell secretory function. Addition of interleukin‐4 (IL‐4) or the nitric oxide synthase inhibitor N‐monomethyl‐l‐arginine to peritoneal cells inhibited nitric oxide synthesis and enhanced anti‐IgE‐mediated mast cell degranulation, measured as serotonin release. Interferon‐γ (IFN‐γ) completely overcame the enhancement of serotonin release and suppression of nitrite production induced by IL‐4. Over several experiments, with or without IL‐4 and/or IFN‐γ, serotonin release correlated inversely with nitrite production. On a cell‐for‐cell basis, non‐mast cells produced ≈30 times more nitrite than mast cells in peritoneal cell populations, with or without IFN‐γ stimulation. The nitric oxide donor S‐nitrosoglutathione inhibited anti‐IgE‐induced serotonin release from purified mast cells, whereas 8‐bromo‐cyclic GMP, the guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one, superoxide dismutase and the peroxynitrite scavenger uric acid, were without effect. We conclude that IL‐4 and IFN‐γ reciprocally regulate mast cell secretory responsiveness via control of nitric oxide synthesis by accessory cells; the nitric oxide effect on mast cells is direct but does not involve cyclic GMP or peroxynitrite.
Genetically susceptible mice remain proportionally more susceptible to tuberculosis after vaccination
Authors: MEDINA  , NORTH
Published: March 2003

DBA/2 mice are much more susceptible to infection with Mycobacterium tuberculosis than major histocompatibility complex‐compatible BALB/c mice. It is shown here that, although vaccination provided mice of both strains with a capacity to reduce the level of infection in their lungs, vaccinated DBA/2 mice remained much more susceptible in this organ than vaccinated BALB/c mice. Consequently, the former mice developed more lung pathology and died much earlier than the latter. On the other hand, colony‐forming unit counts and histology suggest that vaccination provided mice of both strains with an increased and equal ability to express immunity in the liver and spleen, thereby indicating that they possessed equal systemic levels of vaccine‐induced immunity at the time of M. tuberculosis challenge. The results indicate that inefficient expression of immunity in the lungs is likely to prove an obstacle to successful vaccination against tuberculosis in resistant and susceptible mouse strains, but more so in the latter strains.
Extrathymic derivation of gut lymphocytes in parabiotic mice
Authors: SUGAHARA  , SHIMIZU  , YOSHIDA  , AIBA  , YAMAGIWA  , ASAKURA  , ABO
Published: March 2003

In adult mice, c‐kit+ stem cells have recently been found in their liver, intestine and appendix, where extrathymic T cells are generated. A major population of such thymus‐independent subsets among intraepithelial lymphocytes is T‐cell receptor (TCR)γδ+ CD4− CD8αα+(β−) cells, but the origins of other lymphocyte subsets are still controversial. In this study, we examined what type of lymphocyte subsets were produced in situ by such stem cells in the small intestine, large intestine and appendix. To investigate this subject, we used parabiotic B6.Ly5.1 and B5.Ly5.2 mice which shared the same circulation by day 3. The origin of lymphocytes was identified by anti‐Ly5.1 and anti‐Ly5.2 monoclonal antibodies in conjunction with immunofluorescence tests. Lymphocytes in Peyer’s patches and lamina propria lymphocytes (especially B cells and CD4+ T cells) in the small intestine became a half‐and‐half mixture of Ly5.1+ and Ly5.2+ cells in each individual of parabiotic pairs of mice by day 14. However, the mixture was low in CD8αα+, CD8αβ+ and γδ T cells in the small and large intestines and in CD3+ CD8+ B220+ cells in the appendix. These cells might be of the in situ origin. When one individual of a pair was irradiated before parabiosis, the mixture of partner cells was accelerated. However, a low‐mixture group always continued to show a lower mixture pattern than did a high‐mixture group. The present results suggest that extrathymic T cells in the digestive tract may arise from their own pre‐existing precursor cells and remain longer at the corresponding sites.
Antigen‐specific B and T cells in human/mouse radiation chimera following immunization in vivo
Authors: BÖCHER  , MARCUS  , SHAKARCHY  , DEKEL  , SHOUVAL  , GALUN  , REISNER
Published: March 2003

Adoptive transfer of human peripheral blood mononuclear cells (PBMC) into mice with severe combined immunodeficiency (SCID) or into lethally irradiated BALB/c mice radioprotected with SCID bone marrow, leads to marked engraftment of human T and B cells. In such chimeras, human serum antibody responses can be stimulated readily by vaccination with recall antigens, but the detection of antigen‐specific functional T or B cells has been extremely difficult. In the present study, we were able to detect by Elispot analysis high frequencies of immunoglobulin G (IgG)‐secreting B cells and mitogen‐responsive interferon‐γ (IFN‐γ) or interleukin‐4 (IL‐4)‐secreting T cells in peritoneum and spleen of human/BALB/c chimeric mice during the first 3 weeks after PBMC transfer. Moreover, specific memory responses were elicited by vaccination with tetanus toxoid (TT) or hepatitis B virus (HBV) surface (HBs) antigen of chimeric mice transplanted with PBMC derived from TT‐ or HBV‐immune donors. Substantially higher TT‐specific B‐cell frequencies were found during the first 3 weeks after vaccination in mice challenged with the specific antigen compared to the levels found in control animals. High numbers of TT‐specific IFN‐γ‐secreting T cells persisted in the peritoneum of vaccinated, but not of unvaccinated, animals during the entire observation period, but only low numbers of specific IL‐4‐secreting T cells were found in vaccinated mice. Similar results were achieved following vaccination with HBs antigen of chimeric mice, transplanted with PBMC of HBV immunized donors. Thus, TT or HBsAg‐specific antibody responses in our model correlate closely with the existence of specific IFN‐γ‐secreting T helper 1/0 cells. Furthermore, these results show that adoptive transfer of human PBMC into lethally irradiated mice provides an efficient approach to generate specific B‐cell fusion partners for the production of human monoclonal antibodies and specific T‐cell lines for adoptive cell therapy of malignant or infectious diseases.
The lifespan of major histocompatibility complex class I/peptide complexes determines the efficiency of cytotoxic T‐lymphocyte responses
Authors: MICHELETTI  , BAZZARO  , CANELLA  , MARASTONI  , TRANIELLO  , GAVIOLI
Published: March 2003

Major histocompatibility complex (MHC)/peptide association and stability are determined by specific amino acid interactions between peptide antigens and the MHC groove, and are regarded as a critical feature in ensuring efficient monitoring by T cells. In this investigation we examined the relationship between MHC/peptide stability and the immunostimulatory capacity of MHC/peptide complexes. For this purpose we compared synthetic peptide analogues derived from the immunodominant HLA‐A11‐presented IVTDFSVIK (IVT) epitope, for their capacity to reactivate IVT‐specific memory cytotoxic T‐lymphocyte (CTL) responses. The analogues differentiated from the wild‐type epitope by single amino acid substitution at position 2. All peptides showed similar affinity for HLA‐A11 molecules and were recognized by IVT‐specific CTL clones, but induced HLA‐A11 complexes at the cell surface with different lifespan. This model offered the possibility of comparing the capacity of an immunogenic epitope to stimulate a unique population of T‐cell precursors depending on the lifespan of its presentation at the cell surface. We demonstrated that stable HLA‐A11/peptide complexes efficiently stimulate IVT‐specific CTL responses, while HLA‐A11/peptide complexes with short lifespan do not. The precise identification of the role of amino acid residues in the formation of stable MHC/peptide complexes may be relevant for the design of wild‐type‐derived epitopes with high immunogenicity. These analogues may have important applications in the immunotherapy of infectious diseases and immunogenic tumours.
Direct evidence that ERK regulates the production/secretion of interleukin‐2 in PHA/PMA‐stimulated T lymphocytes
Authors: LI  , HII  , DER  , FERRANTE
Published: March 2003

Although p21ras, raf‐1 and MEK have been shown to regulate directly the transcriptional activity of NFAT (nuclear factor of activated T cells) and/or the interleukin‐2 (IL‐2) promoter, direct evidence that the extracellular signal‐regulated protein kinase (ERK) is involved in regulating IL‐2 production is still lacking. Here, we demonstrate that transfection of Jurkat cells with a dominant negative mutant of ERK1 (Erk1‐K71R) resulted in the suppression of mitogen‐stimulated production/secretion of IL‐2. This was accompanied by a parallel inhibition of mitogen‐stimulated ERK activity. These data provide direct evidence, for the first time, that ERK plays a vital role in regulating the production/secretion of IL‐2.
Inhibition of mast cell‐dependent anaphylaxis by sodium salicylate
Authors: KIM  , SHIN  , CHOO  , PARK
Published: March 2003

Sodium salicylate (NaSal) is a commonly used agent with a wide pharmacological spectrum. The objective of the present study was to investigate the effect of NaSal on anaphylaxis. NaSal (10−1and 1 mm) significantly inhibited systemic anaphylaxis induced by compound 48/80 in rats. NaSal also significantly inhibited local anaphylaxis activated by anti‐dinitrophenyl (DNP) immunoglobulin E (IgE). NaSal (10−1and 1 mm) significantly inhibited histamine release from rat peritoneal mast cells (RPMC) activated by compound 48/80 or anti‐DNP IgE. Northern‐blot analysis demonstrated that a significantly reduced level of the mRNA of l‐histidine decarboxylase was expressed in mast cells treated with NaSal, compared with that without NaSal. NaSal (10−2and 10−1 mm) had a significant inhibitory effect on anti‐DNP IgE‐induced tumour necrosis factor‐α secretion from RPMC. The level of cyclic AMP in RPMC, when NaSal (1 mm) was added, transiently and significantly increased about sixfold compared with that of basal cells. These results suggest a possible use of NaSal in managing mast cell‐dependent anaphylaxis.
Defective immune response and severe skin damage following UVB irradiation in interleukin‐6‐deficient mice
Authors: NISHIMURA  , TOHYAMA  , SATOH  , NISHIMURA  , REEVE
Published: March 2003

Interleukin‐6 (IL‐6), a multifunctional cytokine, is induced in the acute‐phase reaction following ultraviolet (UV) irradiation of humans and mice. Using IL‐6‐deficient (IL‐6−/−) mice, we investigated the role of IL‐6 in immunosuppression and inflammatory responses caused by UVB (280–320 nm) radiation. The IL‐6−/− mice had a defective contact hypersensitivity (CHS) in response to the sensitizers 2,4‐dinitrofluorobenzene and oxazolone. The injection of recombinant IL‐6 (rIL‐6) into these mice resulted in a marked recovery of the CHS. Serum IL‐6 was significantly elevated by UV irradiation of wild‐type B6 J/129Sv (IL‐6+/+) mice but was not detectable in IL‐6−/− mice. Interestingly, there was no induction of serum interleukin‐10 (IL‐10) by UV irradiation of IL‐6−/− mice, whereas UV exposure caused a significant increase in serum IL‐10 levels in IL‐6+/+ mice. Injection of rIL‐6 into IL‐6−/− mice increased IL‐10 to levels similar to those of IL‐6+/+ mice. Being different from IL‐6+/+ mice, no epidermal proliferation was found at 48 hr in the IL‐6−/− mice, but delayed cell proliferation was observed at 72 hr after UV exposure. Immunohistochemical analysis demonstrated that the epidermis was capable of synthesizing IL‐6 at 72 hr after UV irradiation of IL‐6+/+ mice. In addition, the IL‐6‐positive cells appeared to be Langerhans’ cells, which were detected with dendritic cell‐reactive S‐100 antibody. The present study strongly suggests that IL‐6 may play a crucial role in the alteration of cutaneous immune responses following UV exposure, and provides evidence that IL‐6 is a potent inducer of IL‐10. Furthermore, IL‐6 production induced by UV radiation appears to be an important early signal for repair of UV‐caused skin damage.
Approach to withdrawal from tacrolimus in a fully allogeneic murine skin graft model
Authors: Uchiyama  , Kong  , Kishihara  , Sugimachi  , Nomoto
Published: March 2003

With few exceptions, transplant patients must take immunosuppressants throughout their lives. In this study, we used anti‐T‐cell receptor (TCR/CD3) monoclonal antibodies (mAbs) to induce immunological tolerance to alloantigens after withdrawal from tacrolimus in a fully allogeneic murine skin graft model. Skin grafts from AKR donor mice were maintained in C57BL/6 recipients by administering tacrolimus for one month. Anti‐T‐cell receptor (TCR) αβ mAb was administered to recipient mice on the day of withdrawal from tacrolimus administration. Seven days after mAb administration, the recipient mice were treated with various combinations of the following treatments: low‐dose whole body irradiation, AKR bone marrow transfer (BMT), and anti‐CD3 mAb administration. The control recipient mice did not receive treatment with either mAb, nor any other treatment. All the control recipient mice showed rejection of AKR skin grafts 42 days after tacrolimus withdrawal (mean skin graft survival: 77 days). Mice treated with a combination of anti‐TCR αβ antibody, low‐dose irradiation and AKR BMT showed stable chimerism in their peripheral blood lymphocytes and significantly prolonged skin graft survival (mean skin graft survival: >151·2±15·3 days). Mice given the combination of anti‐TCR αβ mAb, anti‐CD3 mAb, low‐dose irradiation, and AKR BMT exhibited more stable chimerism but had earlier skin graft rejection (mean skin graft survival: 116·7±17·6 days) than the mice that did not receive anti‐CD3 mAb. These results suggest that anti‐TCR αβ mAb, but not anti‐CD3 mAb, in combination with low‐dose irradiation and BMT, is useful for long‐lasting allograft survival after withdrawal from tacrolimus in mice with fully allogeneic skin grafts.
Interferon‐γ regulates the interaction of RBL‐2H3 cells with fibronectin through production of nitric oxide
Authors: Wills  , Gilchrist  , Befus
Published: March 2003

Interferon‐γ (IFN‐γ) is an important regulatory cytokine in cell proliferation, differentiation, adhesion, mediator release, and gene induction. This diversity of effector roles is achieved by a variety of incompletely understood mechanisms. In the mast cell (MC), IFN‐γ downregulates mediator synthesis and secretion. The present study demonstrates and characterizes for the first time IFN‐γ inhibition of adhesion of the MC analogue RBL‐2H3 to the extracellular matrix protein fibronectin (FN). Inhibition requires preincubation of the cells with IFN‐γ for 20 hr, and is statistically significant at 100 U/ml IFN‐γ. Flow cytometry indicates that cell surface expression of very late antigen‐4 (VLA‐4), VLA‐5, and the vitronectin receptor (VNR) remain constant following IFN‐γ treatment, indicating the inhibitory effect of IFN‐γ on adhesion to FN is not achieved through a reduction in integrin receptors for FN. Fluorescent labelling with Texas red phalloidin demonstrated rearrangement of the actin cytoskeleton in response to IFN‐γ was not significant. The tyrosine phosphatase inhibitor vanadate, and the nitric oxide (NO) synthase inhibitor L‐NAME, reduced the IFN‐γ effect on adhesion to FN by 62 and 70%, respectively, demonstrating that the IFN‐γ effect is dependent upon the production of NO, potentially though a tyrosine phosphatase dependent mechanism. The NO donors sodium nitroprusside and S‐nitrosoglutathione mimicked the effect of IFN‐γ. Thus, following stimulation with IFN‐γ, NO plays an autocrine role in the MC, and is able to modulate integrin function. This adds to the pathways NO is able to inhibit in the mast cell, shows that endogenous NO is able to inhibit these pathways, and suggests NO is impinging upon an element common to many signalling mechanisms in the MC.
Endogenous interleukin‐12 only plays a key pathogenetic role in non‐obese diabetic mouse diabetes during the very early stages of the disease
Authors: Nicoletti  , Di Marco  , Zaccone  , Magro  , Di Mauro  , Grasso  , Meroni
Published: March 2003

A rat monoclonal antibody (mAb) that neutralizes mouse interleukin‐12 (IL‐12) was administered to female non‐obese diabetic (NOD) mice of different ages to dismantle the role of endogenous IL‐12 in murine autoimmune diabetogenesis. This mAb was effective in preventing clinical, but not histological signs of spontaneous diabetes when treatment was started early in life at the age of 4 weeks and consecutively continued for 10 weeks. Delaying commencement of anti‐IL‐12 mAb prophylaxis until the age of 18 weeks, when NOD mice suffer from advanced insulitis, was ineffective. Anti‐IL‐12 mAb did not influence the course of the accelerated model of diabetes induced by cyclophosphamide. These data prove that the pathogenetic role of endogenous IL‐12 in NOD mouse diabetes is restricted to the very early diabetogenic events presumably occurring prior to insulitis development.
Rat complement factor I: molecular cloning, sequencing and expression in tissues and isolated cells
Authors: Schlaf G. , Rothermel E. , Oppermann M. , Schieferdecker H. L. , Jungermann K. , Götze O.
Published: March 2003

Factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the α‐chain of C3b and two bonds in the α‐chain of C4b thereby inactivating these proteins. The human protein and the recently characterized mouse factor I are heterodimers of about 88 000 MW which consist of a non‐catalytic heavy chain of 50 000 MW which is linked to a catalytic light chain of 38 000 MW by a disulphide bond. For the screening of a rat liver cDNA library we used a hybridization probe produced by polymerase chain reaction (PCR) using degenerated primers which corresponded to conserved parts of the human and the murine factor I nucleotide sequences. One of the identified sequences, which had a length of 2243 base pairs (bp), contained the complete coding region and the whole 3′ untranslated region. The length of the coding region in rat consisted of 1812 bp followed by a 3′ untranslated region of 207 bp including the polyadenylation signal and the beginning of the poly A tail. Comparison of the rat cDNA‐derived coding sequence revealed identities of 87% to the mouse and of 78% to the human FI nucleotide sequence. The translation product of rat FI mRNA was 604 amino acid residues (aa) in length with an identity of 85% to the mouse (603 aa) and 69% to the human protein (583 aa). The comparison of the molecular mass predicted by the primary structure and derived from rat FI isolated from rat serum as detected in immunoblot analyses suggested a glycosylation of more than 20% of the total mass of the FI protein. Expression studies using reverse transcription (RT)–PCR assays indicated that FI‐specific mRNA could neither be identified in B cells, nor in T cells, monocytes or granulocytes from rat and human peripheral blood nor in rat peritoneal macrophages. These data were in agreement with the results of RT–PCR obtained with several human lymphoma cell lines (Jurkat, MOLT‐4, HUT102, Wil 2‐NS, Ramos, Raji, U937) all of which were devoid of FI‐specific mRNA. In accord with our data from two rat hepatoma cell lines (FAO and H4IIE) and one from man (HepG2) only isolated rat hepatocytes (HC) but neither Kupffer cells (KC), hepatic stellate cells (HSC; Ito cells) nor sinusoidal endothelial cells (SEC) expressed FI‐specific mRNA. FI mRNA was also detected in human umbilical vein endothelial cells (HUVEC) and in the uterus and small intestine of the rat. Spleen and lymph nodes did not contain any detectable FI‐specific mRNA.
Interleukin‐12 modulates T‐cell responses to microfilariae but fails to abrogate interleukin‐5‐dependent immunity in a mouse model of onchocerciasis
Authors: Hogarth P. J. , Bianco A. E.
Published: March 2003

Infection of mice with microfilariae of Onchocerca lienalis induces high levels of protective immunity to reinfection, which is dependent on interleukin (IL)‐5 but not IL‐4. Here, we have investigated the effect of exogenous IL‐12 administration during either the priming or effector phases of the immune response. When administered during priming, IL‐12 induced down‐regulation of parasite‐specific serum immunoglobulin (Ig)E and up‐regulation of IgG2a. Antigen‐specific IL‐4 responses were strongly suppressed, whilst blood eosinophil levels were partially reduced. When administered during a challenge infection, IL‐12 did not significantly influence the balance of antibody isotypes, but partially reduced eosinophil production. Antigen‐specific IL‐4 responses were again completely ablated. Unusually, interferon‐γ (IFN‐γ) responses were not significantly affected following IL‐12 administration, either during priming or after challenge infections. Moreover, despite a fall in antigen‐specific IL‐5 production, the expression of IL‐5‐dependent immunity, as determined by reduction in worm recoveries, was fully maintained. These data demonstrate that parasite‐induced IL‐4 can be abrogated without affecting protective immunity to Onchocerca microfilariae in mice. In view of the established role of IL‐4 in pathogenesis, this may have important implications for the development of immunoprophylaxis aimed at microfilariae and the alleviation of pathology in onchocerciasis.
Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin‐immunized mice
Authors: Zhang T. , Maekawa Y. , Hanba J. , Dainichi T. , Nashed B. F. , Hisaeda H. , Sakai T , Asao T , Himeno K. , Good R. A. , Katunuma N.
Published: March 2003

We previously reported that CA074, a specific inhibitor of cathepsin B, modulates specific immune responses from the T helper 2 (Th2) type to Th1 type in BALB/c mice infected with Leishmania major. In the present study, we found that a similar type of immune deviation was also induced in mice immunized with ovalbumin (OVA). However, treatment of mice with pepstatin A, a specific cathepsin D inhibitor, suppressed the OVA‐specific proliferation of lymphocytes and blocked the development of both Th1 and Th2 cellular responses. These inhibitors did not appear to have any direct influence in vitro on functions of naive lymphocytes. OVA antigen (47 000 MW) was digested mainly into 40 000 MW protein in vitro by lysosomal proteases from naive BALB/c mice, and its digestion was markedly inhibited by the addition of CA074, but not by addition of pepstatin A, during incubation. However, pepstatin A strongly suppressed the degradation of the major histocompatibility complex class II‐associated invariant chain (Ii) molecule in vivo and in vitro. Thus, cathepsin B appears to process antigens directed to preferential activation of Th2 cells, while cathepsin D may be responsible for the degradation of Ii, the processing of which is essential in initiating the antigen‐specific activation of Th1 and Th2 CD4+ T cells. These lysosomal proteases may have different functions in regulating immune responses.
A monoclonal antibody to the rat Crry/p65 antigen, a complement regulatory membrane protein, stimulates adhesion and proliferation of thymocytes
Authors: Arsenović‐Ranin N. , Vučević D. , Okada N. , Dimitrijević M. , Čolić M.
Published: March 2003

A murine monoclonal antibody (mAb), 3F10, was produced by fusion of spleen cells obtained from mice immunized with a rat cortical thymic epithelial cell line (R‐TNC.1) stimulated with interferon‐γ and P3X myeloma cells. 3F10 recognized an antigen expressed both on thymocytes and non‐lymphoid cells in the thymus. Flow cytometry showed that 3F10 stained more than 98% thymocytes and 90% R‐TNC.1 cells. Immunoprecipitation and Western blot studies demonstrated that 3F10 reacted with molecules of 55000 and 65000 MW from both thymocyte and R‐TNC.1 cell lysates. 3F10 recognized the same antigen on Chinese hamster ovary cells transfected with rat Crry as did 5I2 mAb, confirming the specificity of 3F10 mAb for the rat homologue of mouse Crry/p65, a membrane‐bound complement regulatory protein. 3F10 mAb induced homotypic aggregation of thymocytes and exhibited an additive effect on the aggregation evoked by phorbol myristate acetate. The aggregation was dependent on active cell metabolism, intact cytoskeleton, divalent cations and activation of protein phosphatases 1 and 2A (as assessed by use of okadaic acid). In contrast, H‐7, HA1004 and genistein partially inhibited, whereas staurosporine potentiated the aggregation of thymocytes triggered by 3F10. 3F10 mAb also stimulated binding of thymocytes to the R‐TNC.1 line. Both homotypic and heterotypic adhesive interactions are mediated by leucocyte function‐associated antigen‐1 (LFA‐1). In addition, 3F10 stimulated proliferation of thymocytes induced by suboptimal concentrations of concanavalin A. These data suggest that rat Crry/p65 might be involved in the regulation of both cell adhesion and activation of thymocytes. This is a novel, non‐complement‐dependent function of Crry/p65.
Presence of B220 within thymocytes and its expression on the cell surface during apoptosis
Authors: Oka S. , Mori N. , Matsuyama S. , Takamori Y. , Kubo K.
Published: March 2003

B220 is the full‐length splicing isoform of a tyrosine phosphatase CD45 and is predominantly expressed as a transmembrane protein on B cells. Other splicing isoforms of CD45 are yielded by alternative splicing of exons 4, 5 and 6. Recently, the expression of B220 on peripheral T cells during activation‐induced cell death has been reported. To investigate whether B220 is implicated in apoptosis of immature T cells, we analysed (by flow cytometry using the anti‐B220 monoclonal antibody, RA3‐6B2) the expression of B220 on mouse thymocytes undergoing X‐irradiation‐ and dexamethasone (DEX)‐induced apoptosis. The expression of B220 on thymocytes positive for Thy‐1 was induced by X‐irradiation or DEX treatment and increased with length of incubation. The expression of B220 was pronounced on the apoptotic hypodiploid cells in the fraction showing lower forward scattering values. Reverse transcription–polymerase chain reaction detected mRNA containing exons 4, 5 and 6 of CD45 in normal thymocytes as well as those exposed to X‐rays or DEX. Surprisingly, cytoplasmic B220 antigens were detected in a considerable fraction of normal thymocytes. Moreover, the expression level of the 220 000‐MW protein in normal thymocytes was similar to that in the thymocytes undergoing apoptosis. During apoptosis, the expression level of B220 antigen was reduced in the cytoplasm but, conversely, up‐regulated on the surface of thymocytes. These results suggest that B220 is constitutively expressed as a cytoplasmic form within thymocytes and possibly translocated to the cell membrane during apoptosis.
Specificity and binding kinetics of murine lupus anti‐DNA monoclonal antibodies implicate different stimuli for their production
Authors: Eivazova E. R. , McDonnell J. M. , Sutton B. J. , Staines N. A.
Published: March 2003

The origin and relative biological importance of the many different DNA‐reactive antibodies that appear in systemic lupus erythematosus are not well understood. A detailed analysis of their fine specificity and binding characteristics with DNA is a necessary step in understanding their biology. We have examined here two monoclonal antibodies (mAb) IV‐228 and V‐88 that are, respectively, characteristic of antibodies, which bind exclusively to single‐stranded (ss) DNA and to both double‐stranded (ds) DNA and ssDNA. By surface plasmon resonance (SPR) on BIAcore, we characterized the kinetics of binding of each antibody to synthetic ss and ds oligonucleotides. Antibody V‐88 and IV‐228 showed different patterns of reactivity for both ss and ds oligonucleotides, characterized by distinctly different kinetic parameters. Analysis of their binding kinetics indicates the importance of base composition in defining DNA epitopes, and shows that some epitopes, such as that recognized by mAb V‐88, are expressed on dsDNA and ssDNA, whereas others, as recognized by IV‐228, are not. The base preferences of V‐88 for ds GC‐rich structures over AT‐rich, and of IV‐228 for ss T‐rich structures, also reveal distinct differences between these antibodies. We conclude that the different binding properties of the antibodies will relate to their biological activities. The base preferences of the antibodies suggest that they might be induced by different immunological stimuli, such as those that could be provided by the various DNA fragments and structures released during programmed cell death.
Natural antibody‐induced intracellular signalling and growth control in C3H 10T½ fibroblast variants
Authors: Wang H. , Chow D. A.
Published: March 2003

Cell‐surface binding by natural antibody (NAb) places it well for controlling cell function directly through signalling. Flow cytometry revealed an instability of syngeneic NAb binding to C3H 10T½ fibroblast variants at 37°, which could be partially reduced by H7, an inhibitor of the pivotal signalling serine/threonine kinase, protein kinase C (PKC). Cells coated with purified NAb at 4° followed by a rise in temperature to 37° showed an increase in membrane expression of introduced rat PKC‐β1 and endogenous PKC‐α, in the PKC‐β1‐overexpressing PKC‐4 and v‐H‐ras‐producing I3T2.1, respectively. Tyrosine phosphorylation of membrane‐associated 60 000 MW protein including the tyrosine kinase src was markedly reduced. In addition, both the precoated NAb and numerous membrane molecules ranging from 20 000 to 220 000 MW were released into the supernatant, including the receptor‐like protein tyrosine phosphatase α (RPTP‐α). Furthermore, purified NAb reduced the growth of I3T2.1 cells in culture assessed as a decrease in total cell numbers and an increase in the proportion of cells in the G0/G1 phase of the cell cycle. Together, these data argue that the interaction of NAb with cell surface structures initiated a series of intracellular signalling events leading to the release of membrane molecules and over time the suppression of cell proliferation. This process could provide a biological mechanism for direct NAb control of activated cells in both physiological and pathological conditions.
Role of β1 and β2 subunits of the interleukin‐12 receptor in determining T helper 1/T helper 2 responses in vivo in the rat
Authors: Gillespie K. M. , Szeto C.‐C. , Betin V. M. , Mathieson P. W.
Published: March 2003

Interleukin‐12 (IL‐12) responsiveness, and hence capacity to mount a T helper type 1(Th1) immune response, may be regulated via differential expression of the IL‐12 receptor β2 subunit at least in vitro in human and murine cells. To test whether a similar phenomenon operates in vivo in the rat we cloned and sequenced partial cDNAs for rat IL‐12Rβ1 and IL‐12Rβ2 subunits and analysed expression of these genes in vivo in two rat strains with different Th1/Th2 bias. After treatment with mercuric chloride (HgCl2), Brown–Norway rats develop Th2‐biased autoimmunity whereas Lewis rats do not develop autoimmunity, instead becoming resistant to Th1‐biased diseases to which they are normally susceptible. We report close sequence homology between the segments of the rat IL‐12R genes sequenced and corresponding mouse genes (95·6% and 92% for IL‐12Rβ1 and IL‐12Rβ2, respectively). Both Brown–Norway and Lewis rats express both β1 and β2 subunits of IL‐12 receptor in vivo in spleen; Brown–Norway rats express the β2 subunit at a lower level than Lewis rats. After HgCl2 treatment, IL‐12Rβ1 expression was not altered but there was down‐regulation of IL‐12Rβ2 expression in both strains. We conclude that relative under‐expression of IL‐12Rβ2 by Brown–Norway rats contributes to their Th2 bias, and that down‐regulation of IL‐12Rβ2 after HgCl2 administration in Lewis rats underlies subsequent resistance to induction of Th1‐biased diseases.
Mast cells enhance contraction of three‐dimensional collagen lattices by fibroblasts by cell–cell interaction: role of stem cell factor/c‐kit
Authors: Yamamoto T. , Hartmann K. , Eckes B. , Krieg T.
Published: March 2003

Reorganization of the extracellular matrix is important in many biological and pathophysiological processes, including tissue remodelling, wound healing, or cancer metastasis. The ability of cultured fibroblasts to reorganize and contract three‐dimensional type I collagen gels is regarded as an in vitro model for this process. In tissue fibrosis, complex interactions among fibroblasts, inflammatory cells and the extracellular matrix are taking place. Mast cells have often been discussed to play a role in several fibrotic conditions including scleroderma, scar formation, or wound healing. In this study, we examined the effects of mast cells on contraction of collagen lattices. The results demonstrate that co‐culture of dermal fibroblasts with a human mast cell line (HMC‐1) significantly enhanced contraction of the three‐dimensional collagen lattices, whereas mast cells alone failed to contract the gel. Addition of culture supernatants of mast cells did not enhance the speed of gel contraction, indicating the importance of cell–cell contact. Morphological analysis showed that mast cells were incorporated into the lattices. Histological examination also demonstrated that within the lattices, mast cells were localized in close contact to, or attached to, fibroblasts. As fibroblasts and mast cells are known to attach via stem cell factor (SCF)/c‐kit interaction when co‐cultured in monolayers, we also examined the effect of antibodies against SCF and c‐kit in this system. Addition of both antibodies inhibited gel contraction up to 70%. In contrast, antibodies against interleukin‐4 (IL‐4) and IL‐4 receptor did not affect gel contraction. These results indicate that mast cells enhance fibroblast‐mediated contraction of collagen lattices via direct cell–cell contact, mediated in part by SCF/c‐kit interactions.
Interleukin‐6 regulates the phenotype of the immune response to a tuberculosis subunit vaccine
Authors: Leal Irene S. , Flórido Manuela , Andersen Peter , Appelberg Rui
Published: March 2003

We investigated the role of interleukin‐6 (IL‐6) in the development of the immune response to a subunit vaccine against tuberculosis consisting of the culture filtrate proteins of Mycobacterium tuberculosis emulsified in the adjuvant dimethyldioctadecylammonium bromide (DDA). C57Bl/6 mice immunized with this vaccine developed a strong T helper 1 (Th1) response characterized by an increased production of interferon‐γ (IFN‐γ) secreted by CD4+ T cells. Neutralization of IL‐6 during in vivo priming resulted in marked reduction in the ability of T cells to secrete IFN‐γ and IL‐2 and to proliferate. IL‐6 gene‐disrupted mice primed with the vaccine showed a decrease in the number of IFN‐γ‐producing cells and an increase in IL‐4‐secreting cells as compared to control mice. In contrast, neutralization of IL‐6 during a boost of the vaccine in previously primed mice did not affect the development of IFN‐γ‐producing cells but still increased the number of IL‐4‐producing cells. Our work shows that IL‐6 plays a major role in the priming but not in the later expression of a Th1 response to a tuberculosis vaccine.
Apoptosis and peripheral blood lymphocyte depletion in coeliac disease
Authors: Di Sabatino Antonio , D'Alò Simona , Millimaggi Danilo , Ciccocioppo Rachele , Parroni Raffaella , Sciarra Giuseppe , Cifone Maria Grazia , Corazza Gino Roberto
Published: March 2003

In coeliac disease (CD) immunological abnormalities are not confined to the small bowel and it has been suggested that changes in peripheral blood lymphocytes (PBL), such as lymphopenia and increased T‐cell activation, may predispose to malignant or autoimmune complications of this condition. In the light of the recent findings about the Fas–Fas ligand (FasL) system in regulating lymphocyte homeostasis, the aim of the present study was to investigate peripheral lymphocyte Fas‐mediated apoptosis in CD to establish whether the homeostatic role of apoptosis in peripheral T‐cell selection is maintained. Moreover, because a soluble form of Fas has been described to be functionally implicated in the Fas signalling system, suggesting a relationship between some disorders and soluble Fas function, we measured levels of soluble Fas in sera of coeliac patients and analysed the relationship between these levels and the proportions of apoptotic and Fas+ PBL to further explore the function of the Fas–FasL pathway in this condition. Finally, we evaluated whether the increased prevalence of anticardiolipin antibodies, recently described in CD, could be related to PBL apoptosis in this condition. We demonstrated an increased apoptosis and higher levels of Fas and FasL expression in PBL isolated from untreated coeliac patients when compared to treated coeliac patients and controls. In addition, low levels of soluble Fas and a significant positive correlation between anticardiolipin antibodies and PBL apoptosis were found in untreated CD. Then, our results showed an increased susceptibility of PBL to undergo Fas‐mediated apoptosis in active CD. This increased apoptosis could be responsible for both lymphopenia and immunogenic exposure of phospholipids with subsequent production of autoantibodies.
Receptor‐mediated phagocytosis of rat macrophages is regulated differentially for opsonized particles and non‐opsonized particles containing β‐glucan
Authors: Reichner Jonathan S. , Fitzpatrick Paul A. , Wakshull Eric , Albina Jorge E.
Published: March 2003

Experiments were conducted to test the hypothesis that opsonic and non‐opsonic phagocytic capacities are differentially regulated by resting and wound‐derived macrophages. Furthermore, the phagocytosis of non‐opsonized zymosan and β‐glucan particles was quantified to determine whether cells differentially regulate non‐opsonic lectinophagocytosis in accordance with the carbohydrate composition of the ligand. In that regard, wound macrophages exhibited profound differential regulation in lectinophagocytosis with a seven‐fold increase in phagocytosis of β‐glucan particles following overnight culture but with a relatively modest increase in internalization of mannan‐containing zymosan. Cultured peritoneal macrophages increased uptake of both particles similarly. Upon activation with interferon‐γ/lipopolysaccharide (IFN‐γ/LPS), wound macrophages selectively suppressed β‐glucan ingestion, while phagocytosis of zymosan particles was unaffected. Lectinophagocytosis was decreased in activated peritoneal macrophages regardless of particle composition and was due in part to a nitric oxide‐dependent mechanism which was without a role in regulation of wound macrophage lectinophagocytosis. Overnight culture of wound macrophages suppressed their capacity for opsonic‐dependent phagocytosis independently of activation, whereas suppression of phagocytosis by peritoneal macrophages was activation‐dependent. Regulation of all three phagocytic pathways was achieved distinctly by peritoneal and wound‐derived macrophages, with changes found in the percentage of resident peritoneal macrophages capable of phagocytosis, whereas the phagocytic capacity of wound macrophages was primarily affected by the number of particles ingested by individual cells. Taken together, these findings demonstrate that the differential regulation of phagocytic pathways encompasses the nature of the phagocytic particle, the site from which macrophages are obtained, their response to activating agents and the mechanism through which the cell population alters its phagocytic potential.
Interleukin‐12 stimulation of lymphoproliferative responses in Trypanosoma cruzi infection
Authors: Galvão da Silva Ana Paula , De Almeida Abrahamsohn Ises
Published: March 2003

The cytokine interleukin‐12 (IL‐12) is essential for resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon‐γ (IFN‐γ), a major activator of the parasiticidal effect of macrophages. A less studied property of IL‐12 is its ability to amplify the proliferation of T or natural killer (NK) lymphocytes. We investigated the role of endogenously produced IL‐12 in the maintenance of parasite antigen (T‐Ag)‐specific lymphoproliferative responses during the acute phase of T. cruzi infection. We also studied whether treatment with recombinant IL‐12 (rIL‐12) would stimulate T‐Ag‐specific or concanavalin A (Con A)‐stimulated lymphoproliferation and abrogate the suppression that is characteristic of the acute phase of infection. Production of IL‐12 by spleen‐cell cultures during T. cruzi infection increased in the first days of infection (days 3–5) and decreased as infection progressed beyond day 7. The growth‐promoting activity of endogenous IL‐12 on T‐Ag‐specific proliferation was observed on day 5 of infection. Treatment of cultures with rIL‐12 promoted a significant increase in Con A‐stimulated proliferation by spleen cells from normal or infected mice. Enhanced T‐Ag‐specific proliferation by rIL‐12 was seen in spleen cell cultures from infected mice providing that nitric oxide production was inhibited by treatment with the competitive inhibitor NG‐monomethyl‐l‐arginine (NMMA). Enhancement of proliferation promoted by IL‐12 occurred in the presence of neutralizing anti‐interleukin‐2 (IL‐2) antibody, suggesting that this activity of IL‐12 was partly independent of endogenous IL‐2. Thymidine incorporation levels achieved with rIL‐12 treatment of the cultures were ≈ 50% of those stimulated by rIL‐2 in the same cultures.