Mononuclear phagocytes migrate into the murine cochlea after acoustic trauma.

Acoustic injury results in destruction of hair cells and numerous nonsensory cells of the cochlea. How these injured structures undergo repair is not well understood. This study was designed to examine the cochlea for the presence of mononuclear phagocytes after tissue injury caused by noise damage. We used octave band noise (8--16 kHz) at three levels (106, 112, and 120 dB) for 2 hours and studied the mice at 1, 3, 7, and 14 days after noise exposure to determine how noise affected hearing thresholds, hair cell number, and tissue injury in the cochlea. Furthermore, we assessed the cochlea for presence of inflammation by performing immunohistochemistry for CD45, common leukocyte antigen. We counted the number of CD45(+) cells that were present in the cochlea at the above-mentioned time points after noise. CD45 is present on all bone marrow-derived white blood cells and is not otherwise expressed in the inner ear. We found that, after noise exposure, there is a large increase in CD45(+) cells. These marrow-derived cells are concentrated in the spiral ligament and spiral limbus, areas that are known to be susceptible to acoustic injury. It is possible that this inflammatory response plays a role in propagating cellular damage in these areas. Immunohistochemistry demonstrates that these cochlear cells are derived from the monocyte/macrophage lineage and serve a phagocytic function in the inner ear.

Skin allograft maintenance in a new synchimeric model system of tolerance.

Treatment of mice with a single donor-specific transfusion plus a brief course of anti-CD154 mAb uniformly induces donor-specific transplantation tolerance characterized by the deletion of alloreactive CD8+ T cells. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. To analyze the mechanisms underlying tolerance induction, maintenance, and failure in euthymic mice we created a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. Chimeras were CBA (H-2(k)) mice engrafted with small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2(b)-alloreactive CD8+ T cells maturing in a normal microenvironment. With this system, we studied the maintenance of H-2(b) allografts in tolerized mice. We documented that alloreactive CD8+ T cells deleted during tolerance induction slowly returned toward pretreatment levels. Skin allograft rejection in this system occurred in the context of 1) increasing numbers of alloreactive CD8+ cells; 2) a decline in anti-CD154 mAb concentration to levels too low to inhibit costimulatory functions; and 3) activation of the alloreactive CD8+ T cells during graft rejection following deliberate depletion of regulatory CD4+ T cells. Rejection of healed-in allografts in tolerized mice appears to be a dynamic process dependent on the level of residual costimulation blockade, CD4+ regulatory cells, and activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization.

Estudo comparativo das infecções por Leishmania major e Leishmania amazonensis em camundongos isogênicos CBA / Comparative study of infections with L. major or L. amazonensis CBA mice

A maioria dos trabalhos sobre o modelo murino na leishmaniose tegumentar utiliza diferentes linhagens de camundongos que são resistentes ou susceptíveis a uma determinada espécie de leishmânia ou trata de manipulações da resposta imune tornando camundongos susceptíveis, resistentes a determinada leishmânia, ou tornando resistentes, susceptíveis. No presente estudo avalia-se comparativamente, resistência e susceptibilidade à infecção por leishmânia, utilizando o modelo de infecção de camundongos isogênicos CBA infectados com L. amazonensis (L.a.), para as quais são susceptíveis, e infectados com L. major (L.m.), para as quais são resistentes. Nós comparamos a resposta imune-inflamatória nesses dois grupos através da avaliação do curso da infecção pelo monitoramento do tamanho das lesões e avaliação da quantidade de parasitos em cortes histológicos através de imunohistoquímica. A resposta tissular foi estudada em cortes histológicos das patas e dos linfonodos de drenagem no intervalo de três a 70 dias após a infecção. A produção de IFN-y, IL-4 e IL-10 foi avaliada pelo método ELlSA e a produção de NO pelo método de Griess, em sobrenadantes de culturas de células do linfonodo de drenagem. Os camundongos CBA infectados por L.m. controlam a infecção e curam, enquanto os infectados por L.a. exarcebam a infecção e morrem. Os padrões de resposta tissular no local da infecção e no linfonodo de drenagem são distintos. Nos animais resistentes ocorre inflamação mista com formação de granuloma e fibrose, enquanto nos susceptíveis ocorre reação macrofágica monomórfica, sem granulomas e fibrose. O IFN-y foi predominante produzido pelas células do linfonodo dos animais infectados por L.m., enquanto que os níveis de IL-4 foram mantidos mais alto no grupo de animais infectados por L.a., após o 7º dia de infecção. Os perfis distintos de resposta correspondem a padrões distintos de resposta tissular e estão relacionados...

Differences in immunological response to a T. gondii protein (SAG1) derived peptide between two strains of mice: effect on protection in T. gondii infection.

This study presents the analysis of the immunogenicity, antigenicity and protective effects of a peptide derived from the major surface antigen of Toxoplasma gondii, SAG1. This synthetic peptide carrying three predicted H-2k restricted T cell epitopes was used to immunize mice. The protective effect of the peptide was evaluated in CBA/J and C57BL/6 mice using the decrease in brain cyst load as evidence of protection. Immunization of C57BL/6 mice yielded high antibody titres but had no protective effect after oral challenge. Immunized CBA/J, mice which responded with a lower titre, showed a 35% reduction in cyst burden after oral challenge. Both strains yielded antibodies which recognized the cognate SAG1 protein on immunoblot assay. Using the BIAcore, system, it was shown that at lower titres the CBA/J mouse sera recognized the native SAG1 protein more effectively than the C57BL/6 mouse sera, yielding much higher anti-peptide titres. Lymphoproliferation assays using the peptide experimentally confirmed the predicted T-cell epitopes and showed that they were also recognized by cells of T. gondii infected mice. The anti-peptide subclass analysis suggested a Th1 orientation in CBA/J mice, whereas a Th2 orientation was observed in C57BL/6 mice. Finally, fine analysis of sequences recognized under MHC class I indicated the existence of a T-cell epitope in the H-2k haplotype (CBA/J mice) but not in the H-2b haplotype (C57BL/6 mice). This study provides a structural basis to the understanding of the vaccination response to one of the T. gondii antigens in different strains of mice.

V beta 8.2 transgene expression interferes with development of experimental autoimmune thyroiditis in CBA k/q but not k/k mice.

The thyroiditogenic T cell receptor (TCR) repertoire is not yet well defined in murine experimental autoimmune thyroiditis (EAT). Our recent work has shown that, while V beta 8+ T cells have no major role in EAT induction with mouse thyroglobulin (MTg), V beta 13 may be involved. To examine the effect of skewing the TCR repertoire on EAT development, CBA (H2k) mice were mated with B10.Q mice harboring an ovalbumin-specific V beta 8.2 TCR transgene (trg), and the trg+ mice were backcrossed to CBA. FACS analysis showed that peripheral blood T cells from trg+ mice had about 76 and 90% V beta 8.2+ cells in the CD4+ and CD8+ subsets, respectively, compared with about 15 and 11% in trg- sibs. The transgenic CBA k/k and k/q mice were immunized with MTg and sacrificed 28 days later. In all trg+ mice, anti-MTg titers and T cell proliferative responses to MTg were significantly lowered. However, thyroid infiltration was distinctly different in the two strains of transgenic mice; a significant decrease was seen primarily in k/q, but not k/k, trg+ mice. Thus, skewing the TCR repertoire to overexpress an irrelevant TCR revealed the possession of a less flexible thyroiditogenic TCR repertoire in k/q, but not k/k, mice.

Occurrence of a soluble nonspecific suppressor factor in the serum early after birth.

A nonspecific suppressor factor has been identified in serum of newborn rats and calves. This factor, designated SUF-s, was shown to interfere--across species barriers--with lymphocyte responses in vitro and in vivo. SUF-s interferes in vitro with T- and B-cell proliferation induced by different mitogens and IL-2. Our findings indicate that the activity of SUF-s in vitro, which is of a reversible nature, is directed at an early event in the cascade of T-cell activation. SUF-s does not affect intrinsically regulated proliferation, such as that of tumor cells or established cell lines. In vivo, SUF-s prevents graft-vs-host disease induced by transplantation of allogeneic bone marrow cells in lethally irradiated mice. Using of affinity chromatography, hydrophobic interaction chromatography, and gel filtration, a 15,000-fold purification of the suppressive factor was attained. The moiety engaged in suppression was identified as a 20- to 40-kDa protein. The biological activity is destroyed at temperatures above 70 degrees C, by proteolytic enzyme digestion and under alkaline conditions but was resistant to acidic and reducing conditions. Judged by its biological activity and some of its physical properties, SUF-s is most likely distinct from other described suppressor factors or known cytokines with suppressor activity, such as IL-4, IL-10, interferon-gamma, transforming growth factor-beta or alpha-fetoprotein.

Anti-Ia treatment prevents lupus-like autoimmune syndrome in mice neonatally tolerized to alloantigens.

Neonatal injection of semi-allogeneic F1 spleen cells into newborn parental mice results in induction of tolerance to the corresponding class I alloantigen and chimerism. This state of tolerance is associated with the development of a transient lupus-like autoimmune syndrome. Previous experiments performed in our laboratories have shown that host CD4+ T lymphocytes and donor B cells persist in the host and are essential in triggering the autoimmune syndrome observed in neonatally tolerized mice. In this study, we show that early treatment of tolerized mice with anti-donor MHC class II mAb totally prevents the lupus-like syndrome. Moreover, delayed treatment significantly decreases, but to a lesser extent, autoimmune pathological features in tolerized mice. Taken together, these results show that lupus-like autoimmune syndrome developed by neonatally tolerized mice is efficiently prevented by anti-Ia treatment without interfering with the induction of tolerance.

B cell developmental defects in X-linked immunodeficiency.

We describe an analysis of early B cell development in mice with X linked immunodeficiency (Xid). It was found that, compared with the normal CBA/J strain, CBA/N (Xid/Xid) pre-B cells show an increased proliferative response to IL-7 but a decreased ability for subsequent maturation on stromal cells. However, the addition of mast cell growth factor largely restored the ability to mature in the presence of stromal cells. No anomalies were found in the rate of Ig heavy or light chain gene rearrangement in CBA/N cells despite their failure to undergo maturation. This suggests that these two events may occur independently.

Dendritic cells are potent antigen-presenting cells for microbial superantigen.

Dendritic cells (DC) were found to be more efficient than macrophages (MO) in activating T cell responses to Staphylococcal enterotoxin B (SEB) using the hanging drop techniques and DC as antigen presenting cells (APC). When superantigen was presented via DC, the activation of T cells was not dependent on antigen processing and MHC class II molecules IA and IE were involved.

Adjuvant action of cholera toxin and pertussis toxin in the induction of IgA antibody response to orally administered antigen.

The ability of cholera toxin B-subunit to bind to intestinal epithelium and in particular the dome epithelium of the Peyer's patch can account for its potency as an immunogen. However, pure B-subunit is a less effective immunogen than whole cholera toxin. The immunogenicity of B-subunit may be restored by the addition of either traces of whole toxin or by pertussis toxin. Cholera toxin and pertussis toxin were both able to stimulate a response when fed in conjunction with keyhole limpet haemocyanin, whereas recombinant B- subunit of heat-labile toxin from Escherichia coli had no effect, demonstrating that the adjuvant action is a property of the enzymically active A-subunits. The adjuvant activity of both pertussis toxin and cholera toxin may be due to their ability to cause an increase in the activity of adenylate cyclase via their action on GTP-binding regulatory proteins. However, feeding of forskolin, a direct activator of adenylate cyclase, had no effect on the mucosal immune response, indicating a role for cholera and pertussis toxin which is independent of enhancement of adenylate cyclase activity in the regulation of the immune response. Antibody to pertussis toxin was not detected, which was attributed to inadequate absorption of pertussis toxin.

Sensitization of allo-specific T lymphocytes in vivo: role of antigen-presenting cells.

The migratory behavior of antigen-presenting cells was investigated in vivo. Purified murine splenic dendritic cells and splenic and peritoneal macrophages were labelled and injected subcutaneously in the hind foot-pads of mice and monitored for seven days. In the first 24 h, a small quantity of label was recovered from popliteal but not inguinal lymph nodes with radioactive (111In-oxine and 3H-uridine) but not fluorescent (1,1'-dioctadecyl 3,3,3'3'-tetramethylindocarbocyanine perchlorate and fluorescein isothiocyanate) labelling of the antigen-presenting cells. Chemical fixation of the injected antigen-presenting cells had no effect on the detection of label in the popliteal lymph nodes, suggesting that it was unlikely to be due to active cellular migration. Label recovery from hind feet declined with time over the seven day period and was independent of the label type. Essentially the same observations were made whether the antigen-presenting cells were syngeneic or allogeneic to the injected mice and irrespective of the type of antigen-presenting cell used. However, allogeneic antigen-presenting cells, which did not migrate to the draining lymph nodes, successfully primed T lymphocytes in these lymph nodes as shown by a secondary in vitro mixed leukocyte reaction. Again, chemical fixation of the injected antigen-presenting cells had no effect on their ability to prime allogeneic T lymphocytes in the draining lymph nodes. These experiments suggest that, during experimental allo-sensitization via the subcutaneous route, indirect priming of allogeneic T lymphocytes may be a dominant pathway.

On the origin of natural IgM in immunoglobulin transgenic mice.

Surface transgenic IgM was expressed by > 95% of small resting splenic B cells but only by 50% of CD5+ and CD5- peritoneal B cells from the mu-transgenic mouse line M54. Transgenic male M54 were crossed with female CBA/N mice carrying the Xid defect. Offspring F1 animals carrying the transgene were analysed for the presence of transgenic and endogenous IgM expressed both in the serum as well as on the surface of splenic and peritoneal B cells. We found that the levels of serum IgM coded for by the transgene were similar in both F1 male, which lack CD5 B cells, and female transgenic mice, which have CD5 B cells. Thus, the Xid defect does not influence the expression of the transgene at the level of naturally activated plasma cells, a finding substantiated by the fact that both male and female naturally activated splenic plasma cells express the transgene at the same frequency. F1 hybrid mice, like transgenic C57BI/6 M54 mice, have naturally activated splenic plasma cells that overexpress endogenous IgM coded for by the VH gene family Q52. The data indicate that normal serum IgM is not derived from CD5+ B cells and that the serum IgM coded for by the mu-transgene from M54 is produced at normal levels even in the male F1 mouse which lacks CD5+ B cells.

V beta 17 T-cell deletion by endogenous mammary tumor virus in wild-type-derived mouse strain.

The wild-type-derived mouse strain PWK possesses a beta-chain variable region V beta 17a2 allele, which is expressed on mature T cells as part of the T-cell receptor of most mice expressing I-E, whereas V beta 17 T cells are deleted in all I-E+ laboratory mice bearing a V beta 17a1 allele. However, (PWK x CBA/J)F1 progeny and the wild-type-derived mouse strain MAI, which possesses the V beta 17a2 allele, display deletion of V beta 17 T cells. Analysis of (PWK x CBA/J) x PWK and of (PWK x MAI) x PWK backcrosses demonstrates that endogenous mouse mammary tumor virus MTV-6 from CBA/J and a MTV from strain MAI control the clonal deletion of V beta 17a2 as well as V beta 3 T cells. Furthermore, among I-E- progeny of a (MAI x C57BL/6) x C57BL/6 backcross, we observed that mice inheriting MTV of MAI have a reduced level of V beta 17 T cells, suggesting that the clonal deletion of V beta 17a2 T cells can be mediated in the absence of the I-E molecule. The 3' long terminal repeat of MTV MAI was cloned and translation of the open reading frame was compared to those of MTV known to encode superantigens. Comparisons indicate that MTV MAI has significantly diverged from the other MTVs. However, MTV MAI and MTV-6 share a stretch of 11 identical amino acids at the C terminus, which is divergent in MTV reacting with other V beta s. This suggests that this region is involved in determining the specificity toward V beta s and has been selectively conserved through evolution of the Mus species.

[Characteristics of immunomodulating effect of histamine in inbred strain mice]. / Osobennosti immunomoduliruiushchego deistviia gistamina u myshei inbrednykh linii.

The activity of 5'-nucleotidase of peritoneal exudate in subcutaneous injection of histamine in a dose of 1.0-100 microliters was studied in mice of different lines (CBA, C57, B1/6, Balb/c, NFS/n, NFR/n). There were interline differences in the influence of histamine on this metabolic index.

In vivo retroviral marking of antigen-specific B lymphocytes.

A major obstacle in the study of B lymphocyte development subsequent to immunization has been the paucity of distinctive phenotypic markers which might permit the separation and scrutiny of a mature antigen-experienced or memory cell population. To address this issue, we have introduced a recombinant non-replicating retrovirus into antigen-reactive lymph nodes in vivo using the expression of a reporter gene to monitor the trafficking, phenotype and persistence of retrovirus-marked B cells. Marked cells are first observed in the medullary cords where they proliferate for approximately 10 days prior to migration to the spleen. Up to half of the marked cells bear antigen-specific immunoglobulin receptors, but otherwise phenotypically resemble conventional B cells. The abundance and persistence of the marked cells suggest an association with the memory compartment. These experiments demonstrate a feasible approach for the identification and lineage analysis of antigen-responsive B cells, and suggest that the lymph node medulla supports a transient phase of post-antigenic B cell development associated with a long-lived antigen-specific B cell subset.

Inter-mouse strain differences in the in vivo anti-CD3 induced cytokine release.

Triggering of the CD3 molecule by in vivo injection of the hamster anti-murine CD3 monoclonal antibody 145-2C11 in adult BALB/c mice leads to massive although transient T cell activation. High levels of tumour necrosis factor (TNF), interferon-gamma (IFN-gamma), IL-2, IL-3 and IL-6 are released into the circulation 1 to 8 h after a single 10 micrograms 145-2C11 i.v. injection. This release induces an impressive self-limited physical reaction associating hypothermia, hypomotility (as assessed by actimetry), diarrhoea, piloerection and even death when high doses (a single dose of greater than 100 micrograms/mouse injection) are administered. In vivo injection of 145-2C11 to other selected mouse strains, namely NZW, CBA/J and C3H/HeJ, induced both different cytokine release patterns and sickness. 145-2C11 induced significant release of TNF and IL-2 in all four strains. At variance, IFN-gamma was only detected in BALB/c mice sera which, in terms of physical reaction (hypothermia and hypomotility) were the most affected. Higher and long-lasting circulating IL-3/GM-CSF levels were present in CBA/J sera, correlating with a later recovery. These results underline heterogeneity in the in vivo cell activation pattern among different mouse strains, when triggering T lymphocytes via the CD3/Ti molecule as compared to exclusive targeting of monocyte/macrophages by means of lipopolysaccharide.

Outgrowth of stable class I major histocompatibility complex-expressing subsets from immunogenic variants of a murine mammary carcinoma: association with a differentially staining region on chromosome 9.

We have examined interactions among intratumor subpopulations during the rejection of immunogenic variants of a murine mammary carcinoma (SPI) and in the outgrowth of tumorigenic "revertant" subsets. Analysis of subclones isolated during the early phase of rejection of one immunogenic variant revealed extensive cellular heterogeneity of tumor-forming ability and class I major histocompatibility complex (MHC) expression. Two main categories of subclones were identified. One set expressed high levels of class I MHC (MHCH) and grew poorly or not at all in normal syngeneic mice. The second set of clones expressed generally low levels of class I MHC (MHCL) and exhibited progressive growth in vivo, similar to the parent tumor. The steady-state mRNA levels for class I MHC and beta 2-microglobulin were constitutively elevated in MHCH clones compared to MHCL clones or the parent tumor. However, in vivo tumorigenic outgrowths from immunogenic variants always expressed the MHCH phenotype. A cytogenetic analysis was carried out to determine the clonal origin and lineage relationship of in vivo selected tumor outgrowths. Surprisingly, tumor outgrowths from mixtures of karyotypically distinct MHCH and MHCL subclones were derived from one lineage within the MHCH subset, despite the fact that MHCH subclones exhibited slower growth in vivo than MHCL subsets when analyzed individually. These results suggest that in polyclonal populations the various subsets sometimes interact in a way that overrides the influence of immunogenic and MHC phenotypes of individual subclones.

Autoimmune response induction and regulation in rat erythrocyte-immunized mice.

The process of antibody formation to self-red blood cells (RBC) has been studied in rat RBC (rRBC)-immunized mice. A positive correlation was noted between antibody production to mouse RBC (mRBC) and rRBC in some mouse strains. The low responsiveness on both indices was overcome by s.c. injections of rRBC in low doses. rRBC-tolerant mice exhibited lower levels of antibody production to mRBC. Splenocytes from rRBC-immunized donors, when transferred to irradiated recipients, revealed enhanced and accelerated anti-mRBC and anti-rRBC antibody production in response to rRBC but not to autologous mRBC. Consequently, the autoimmune process is not accompanied by disordered immunologic tolerance to self-RBC and requires participation of Th responding to foreign epitopes of rRBC antigens. Splenocytes from rRBC-immunized donors, when transferred to non-irradiated recipients, inhibited antibody production to mRBC. The suppressive effect was not abrogated by pretreating donors or recipients with low doses of cyclophosphamide (CP) or by pretreating donors with antibodies to I-J. It was abrogated by the elimination of cells of donor origin within 8-9 days after transfer. Inoculation of antibodies to rRBC in immunized mice on a schedule imitating their splenocyte transfer dynamics inhibited antibody production to mRBC. Therefore, it can be assumed that the suppressive effect of cell transfer is accounted for, not by suppressors or their inducers, but by antibodies to rRBC on the basis of feedback regulation.

Impaired interleukin 2 production by spleen cells from mice infected with human adenovirus.

Spleen cells from mice infected with human adenovirus type 6 (Ad6) showed defective interleukin 2 (IL2) production 3-5 days after the infection. The response of spleen cells to exogenous IL 2 was also deficient. The impaired capacity of concanavalin A--(Con A)-activated spleen cells from Ad6--infected mice to utilize IL 2 seemed to be related to the depressed capacity of the infected splenocytes to express IL 2 receptors. The immunologic dysfunction following infection with Ad6 may be a consequence of deficiencies in both the elaboration of and response to IL 2.