In vivo behavior of peptide-specific T cells during mucosal tolerance induction: antigen introduced through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell priming followed by anergy.

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11. 10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.

Bactericidal activity and postantibiotic effect of levofloxacin against anaerobes.

The bactericidal activity and postantibiotic effect (PAE) of levofloxacin against nine anaerobes were determined. Levofloxacin at concentrations of the MIC and twice the MIC was bactericidal at 24 h to five of nine and nine of nine strains, respectively. The PAE of levofloxacin following a 2-h exposure ranged from 0.06 to 2.88 h.

Interleukin-3 and granulocyte-macrophage colony-stimulating factor enhance the generation and function of dendritic cells.

Dendritic cells, well-known for their potent antigen-presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allo-stimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin-peptide specific class I- and class II-restricted hybridomas containing the lacZ reporter gene, we found that these cytokine-generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)-presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine-generated DC. These findings indicate that exposure in vivo to the cytokines IL-3 and GM-CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of cross-priming and the subsequent generation of tumour-reactive cytotoxic T lymphocytes (CTL).

In-vitro activity of a new oral streptogramin, RPR 106972, alone and in combination with rifampicin or ciprofloxacin against Legionella spp.

The in-vitro activity of RPR 106972, a new oral streptogramin, was compared with that of erythromycin, ciprofloxacin, and rifampicin against 45 Legionella spp. While rifampicin was the most active of all agents tested, RPR 106972 demonstrated activity comparable to that of erythromycin and ciprofloxacin. Usually, indifference was seen when RPR 106972 was tested in combination with rifampicin or ciprofloxacin.

Peptide-specific T cell clonal expansion in vivo following immunization in the eye, an immune-privileged site.

To visualize the primary antigen-specific T cell response to Ag introduced into the eye, we have used an adoptive transfer system in which a limiting number of OVA peptide (323-339)-specific T cells from a TCR-transgenic mouse were transferred into nonirradiated, syngeneic recipients and then tracked in vivo by staining for FACS analysis or immunohistochemistry with the clonotypic mAb KJ1-26. Following posterior chamber injection of Ag, KJ1-26+ cells accumulated primarily in the draining, submandibular lymph node (LN) within 3 days. Although reduced in number, by day 6 these cells were primarily in the paracortical regions and were able to proliferate and secrete IL-2 in response to Ag stimulation. In contrast, following i.v. injection of Ag, the KJ1-26+ cells accumulated in the paracortical regions of the LN to a comparable degree, but did not proliferate or secrete IL-2. The day 3 accumulation of KJ1-26+ cells in the submandibular LN was inhibited if the eye was removed within 5 h after injection of Ag. In the spleen, foci of KJ1-26+ cells were observed in the periarteriolar lymphoid sheaths at day 3; these were not observed to the same degree following other forms of immunization. These results demonstrate that the submandibular LN is the primary site for early clonal expansion of antigen-specific T cells following intraocular Ag administration and that these cells show changes consistent with immunity rather than tolerance.

Transgenic expression of IFN-gamma in the murine lens results in multiple ocular abnormalities and an early but self-limited inflammatory response.

The anterior chamber of the eye is known to be an immune privileged site, due to both local and systemic effects on the immune response. Injection of IFN-gamma into the anterior chamber (AC) overcomes the suppression of antigen-specific delayed hypersensitivity responses normally seen in the eye. Transgenic mice expressing increased IFN-gamma in the lens under the alpha A-crystallin promoter were produced to determine whether the proinflammatory effects of IFN-gamma would abolish immune privilege and promote loss of tolerance as has been seen in non-immune privileged tissues. Two alpha C/IFN-gamma transgenic lines are described which demonstrate multiple ocular and lenticular abnormalities some of which are developmental in origin and others that may be secondary to the inflammatory effects of IFN-gamma. A significant inflammatory cell infiltrate which is observed in the AC and vitreous from birth to 4 weeks of age, consists initially of macrophage and polymorphonuclear leukocytes and then CD4+ T lymphocytes. However, the infiltrate is essentially resolved by 6 weeks of age. Therefore, although lens-specific expression of IFN-gamma results in early loss of immune privilege, chronic uveitis does not occur probably due to the lack of continued IFN-gamma expression.

Characterization of human T-cell responses to Yersinia enterocolitica superantigen.

We reported that antigenic preparations from Yersinia enterocolitica stimulate murine T cells in a manner consistent with that of superantigens. As a consequence we examined whether Y. enterocolitica antigenic preparations stimulate human T-cell cultures. Human T cells, enriched from peripheral blood lymphocytes, were stimulated to proliferate in the presence of Y. enterocolitica cytoplasmic and membrane preparations. This activity has also been shown to be sensitive to protease treatment, indicating the presence of a protein, and when separated by ion-exchange chromatography a single peak of activity is resolved. Furthermore, this proliferation was inhibited, in a dose-dependent manner, by the presence of antibodies directed against MHC class II antigens, indicating a requirement for these molecules. When these cells were stained with a panel of V beta-specific antibodies to determine if there was an enrichment of a particular V beta-bearing T-cell subset after stimulation, results indicate a significant enrichment of T cells bearing V beta 3, V beta 12, V beta 14, and V beta 17 over controls. Taken together, these data are consistent with a Y. enterocolitica product acting as a superantigen for human T cells.

Lens-specific expression of a major histocompatibility complex class I molecule disrupts normal lens development and induces cataracts in transgenic mice.

PURPOSE: Lens epithelial tissue does not normally express major histocompatibility complex (MHC) class I molecules. In addition, the mechanism of self-tolerance to intraocular antigens is unknown. To study the effect of class I expression in the lens, transgenic mice were produced that express an allo-MHC class I molecule under the alpha A-crystallin proximal promoter. METHODS: p alpha Dd was generated by fusion of the H-2Dd structural gene to the alpha A-crystallin proximal promoter. Transgenic mice were produced, and founder lines were identified by Southern blot hybridization. Eyes from transgenic mice were cryostat sectioned and stained for Dd expression or fixed in paraformaldehyde and stained for histologic analysis. Lens RNA was isolated by acid phenol extraction, and transgene expression was analyzed by nuclease protection. RESULTS: The transgenic mice demonstrated dose-dependent, nonimmunologic lens defects consistent within a given line. In the highest expressing lines, ocular defects, including microphthalmia and cataract formation, were observed. Many adult mice from these lines demonstrated lens capsule rupture and a Dd-specific inflammatory response. Inflammation did not occur in mice with intact lens capsules. CONCLUSIONS: Overexpression of H-2Dd in the lens had serious nonimmunologic consequences on lens development and cataract formation. In addition, the high copy number mice revealed at least a partial loss of immunologic tolerance on lens capsule rupture. The lack of an inflammatory response in transgenic mice with intact lens capsules suggests that the physical barrier of the lens capsule is one mechanism of maintaining immune privilege.

Transcription of the murine class II Eb gene is regulated primarily at the level of transcriptional initiation.

The constitutive and inducible expression of MHC class II genes is known to be regulated by multiple upstream promoter elements. Transient transfection assays implicate the proximal promoter region as both necessary and sufficient for appropriate tissue-specific and inducible expression. However, transgenic mouse experiments suggest that additional control regions are important. In this study we tested the hypothesis that additional regulation of class II expression occurred at the level of RNA polymerase II elongation. Nuclear run-on analysis using single-stranded probes spanning the entire Eb gene was performed on a variety of class II-positive, class II-negative, and class II-inducible cell lines. The results demonstrate that, while there is not an even distribution of RNA polymerase along the gene, there is no evidence for a regulated block in elongation. These data further support the idea that the primary mechanism of class II gene regulation is at the level of transcriptional initiation.

Characterization of MHC induction by Mycoplasma fermentans (incognitus strain).

Mycoplasma fermentans (incognitus strain) is a recently identified new human pathogen and suspected cofactor in acquired immune deficiency syndrome. Because this organism appears to exert strong immunosuppressive properties of its own, we decided to investigate whether it was capable of inducing MHC class II expression, as we have observed for other species of mycoplasma. In this report we demonstrate that M. fermentans (incognitus strain) is capable of producing factors that increase MHC class II expression as well as MHC class I expression on the myelomonocytic cell line, WEHI-3 cells. We also present data showing that these mycoplasmal factors induce small, although significant, increases in MHC class I and II antigens on a mouse glioma cell line, G26-20, and MHC class II expression on the human monocyte cell lines, U-937 and HL-60. Using nuclear run-on analysis, we show that the mycoplasma-induced increase in MHC expression is at least partially due to an increase in transcription of the MHC genes. Furthermore, we show that the factor that mediates this activity is sensitive to protease treatment, indicating that it is, at least in part, protein. These results demonstrate that M. fermentans (incognitus strain) is capable of modulating the expression of immunologically important MHC genes in both murine and human cell lines, which may prove to be an important factor in the pathogenesis of this organism.

Two regions of the H-2 Dd promoter are responsive to dimethylsulfoxide in line 1 cells by a mechanism distinct from IFN-gamma.

The line 1 lung carcinoma is a spontaneous BALB/c tumor deficient in class I Ag expression at the protein and mRNA levels. Exposure of line 1 cells to 3% DMSO or IFN-gamma increases class I Ag protein and mRNA dramatically. We have examined the regulation of class I Ag induction by DMSO in line 1 cells. We found DMSO induces class I Ag expression in line 1 cells by a mechanism distinct from IFN, because the kinetics of class I Ag induction by these agents were dramatically different, 7 days vs 3 days, and DMSO did not act through an IFN second messenger. At the molecular level, class I H chain transcription in line 1 cells was low. Treatment with 3% DMSO or IFN-gamma increased H chain transcription four-fold and sevenfold, respectively, indicating that class I H chain expression is regulated at the level of transcription in line 1 cells. Using reporter gene constructs, we mapped the regions in the Dd H chain promoter that increase H chain expression after DMSO treatment of line 1 cells. Two regions of the Dd promoter, D1, from -210 to -133 bp, and D2, from -125 to -61 bp, were found to be independently responsive to DMSO. These regions were also responsive to IFN-gamma in line 1 cells. However, consistent with our cellular results, DMSO and IFN induction of class I H chain expression differed at the molecular level as determined by D1 point mutations that diminished IFN-gamma responsiveness but did not alter induction by DMSO. Thus, DMSO appears to regulate class I transcription through multiple regions of the class I H chain promoter in line 1 cells by a mechanism distinct from IFN-gamma.

Yersinia enterocolitica produces superantigenic activity.

We have recently observed that antigenic preparations from Yersinia enterocolitica are capable of inducing strong proliferative responses in normal murine spleen cell cultures. As a consequence of this observation, we evaluated whether Yersinia-derived Ag possess superantigenic activity. Stimulatory activity can be found in culture supernatants, as well as membrane and cytoplasmic fractions of Y. enterocolitica. Cell depletion studies indicate that the primary responding cell is a CD4+ T cell, which requires the presence of APC for responsiveness to Y. enterocolitica Ag. Furthermore, these APC must express MHC class II Ag, as evidenced by the fact that either antibody depletion of class II+ APC or addition of anti-class II antibodies (that block class II Ag on the surface of APC) eliminates the proliferative response. Evaluation of TCR usage by BALB/c T cells responsive to Y. enterocolitica revealed that those T cells bearing V beta 3, 6, and 11 and possibly 7 and 9 were expanded after exposure to Y. enterocolitica Ag preparations. By using a panel of T cell hybridomas, we have shown that hybridomas bearing V beta 3, 7, 8.1, 9, and 11 but not 2, 8.2, 8.3, and 13 respond to Yersinia. When cytoplasmic fractions of Y. enterocolitica were subjected to column chromatography, proliferative activity was enriched approximately 27-fold, and the elution characteristics of the active material suggest that it possesses hydrophobic regions and is, therefore, probably membrane associated. These data indicate that Y. enterocolitica produces antigenic material that has properties consistent with those of T cell superantigens.

The same CCAAT box-binding factor binds to the promoter of two coordinately regulated major histocompatibility complex class II genes.

Using competition mobility shift, methylation interference, and proteolytic clipping DNA binding assays, we demonstrate that the protein binding the major histocompatibility complex A beta CCAAT box is indistinguishable from the protein previously named NF-Y, which binds the major histocompatibility complex E alpha CCAAT box. Although the two CCAAT boxes share the same 10-base core sequence, termed the Y box, their flanking sequences, known to be important for binding, are very different.

Differential induction of bone marrow macrophage proliferation by mycoplasmas involves granulocyte-macrophage colony-stimulating factor.

We have studied the ability of three different Mycoplasma species to induce proliferation of bone marrow-derived macrophages (BMM). We observed a significant mitogenic effect when BMM cells from BALB/c, DBA/2J, SJL, and C57BL/6 mice were incubated with membranes derived from Mycoplasma arginini or M. arthritidis but not when they were incubated with an equivalent amount of M. pulmonis membrane. We also determined that pretreatment of mycoplasma membrane preparations with papain eliminated the ability of these preparations to induce BMM proliferation. To determine whether these membrane fractions acted indirectly by stimulating the production of soluble factors known to stimulate proliferation of BMM cells, we performed blocking studies with antibodies directed against colony-stimulating factor 1 (CSF-1), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor. Our results indicate that antibodies directed against either CSF-1 or IL-3 failed to block mycoplasma-initiated proliferation of BMM cells. However, when anti-GM-CSF was added to proliferative cultures at the time of initiation, we saw a dose-dependent reduction of mycoplasma-initiated proliferation. We conclude that the ability of mycoplasma membranes to initiate the proliferation of BMM is not shared by all species of mycoplasma and that it involves the production of GM-CSF by an as yet undetermined cell.

Negative trans-acting factors extinguish Ia expression in B cell-L 929 somatic cell hybrids.

Numerous studies have implicated trans-acting factors in the regulation of MHC class II gene expression. Some of these factors have been shown to act by inducing the expression of class II genes while others have been demonstrated to downregulate such expression. These reports have dealt almost exclusively with the role of trans-acting factors in the regulation of class II gene expression in hematopoietic-derived cells. We decided to extend these studies to the role trans-acting factors play in nonhematopoietic-derived (NHD) cells. In order to address this question we made somatic cell hybrids between the NHD Ltk- cell line and normal B cells to determine if the existence of positive trans-acting factors from the B cell would lead to the expression of Ltk- class II genes in the resultant hybrid. Our results clearly indicate that not only was there no induction of Ltk- class II gene expression in the hybrids, but there was a loss of B cell class II gene expression as well. These results suggest that Ltk- cells possess negative trans-acting factors that appear to predominate over the positive trans-acting factors possessed by B cells. We have further extended these studies to test the MHC-inducing activity of IFN-gamma and IL-4 on these hybrids. Our results indicate that the hybrids responded to IFN-gamma with an increase in class I but not class II expression for both fusion partners. Furthermore, neither B cell nor L cell class II genes were induced by IL-4. Taken together, these results indicate that Ltk- cells possess negative trans-acting factors that not only maintain the Ia- phenotype of these cells, but also block the action of positive trans-acting factors from B cells.

MHC class II transcription in different mouse cell types. Differential requirement for protein synthesis between B cells and macrophages.

Although the MHC class II genes are known to be regulated transcriptionally, the relative rates of transcription of the four classical class II genes in different cell types have not been investigated. Using nuclear transcriptional analysis, we have investigated the transcriptional rates of the class II genes in the macrophage cell line WEHI-3, normal bone marrow-derived macrophages, L-929 cells, and two different B cell lymphoma lines. Kinetic analysis of class II transcription in IFN-gamma-treated WEHI-3 cells revealed a 4-h delay, followed by a rapid increase in transcription over the next 20 h. A significant basal level of class II transcription, apparent in bone marrow derived macrophages, was also further enhanced by IFN-gamma treatment. None of the class II genes were transcribed in L cells, whereas all class II genes were transcribed constitutively in the B cell lines. In both B cell lines and macrophages, the four class II genes were found to be transcribed at different rates from one another, but the only gene showing a consistent pattern in multiple experiments was A-alpha, always showing the highest rate. We also investigated the effect of protein synthesis inhibition on class II transcription. Cycloheximide treatment of WEHI-3 cells did not inhibit IFN-gamma-induced transcription of the class II genes within 8 h, suggesting that IFN-gamma acts on pre-existing trans-acting factors, rather than inducing their synthesis. In contrast, treatment of B cells with cycloheximide for 8 h significantly reduced class II transcription, suggesting that, in B cells, continuous synthesis of a labile trans-acting factor is required for constitutive expression. These data support the notion that class II expression in B cells is mediated by trans-acting factors distinct from those found in macrophages.

Induction of class II MHC antigen expression in macrophages by Mycoplasma species.

Several different Mycoplasma species have been shown to act as mitogens for either T or B cells and as stimulators of macrophage tumoricidal activity. In this report, we show that at least five different species of Mycoplasma are capable of inducing class II MHC expression on macrophages. We have observed significant induction of class II MHC surface expression on the myelomonocytic cell line, WEHI-3, as early as 24 h after deliberate infection of cultures, reaching maximal levels by 4 days. This induction was also apparent at the mRNA level as assessed by Northern blot analysis by using A alpha, E alpha, and A beta probes. However, unlike many other previously described MHC-inducing agents, mycoplasmas failed to induce class I MHC expression at either the cell surface or mRNA levels. Kinetic analysis revealed that induction of class II mRNA by mycoplasmas was slower than induction by IFN-gamma requiring 24 h rather than 8 h for significant increases to be noted. Induction by mycoplasmas does not require the presence of live organisms and remains active after heat treatment of 90 degrees C for 30 min. We have also demonstrated that mycoplasma infection of primary bone marrow macrophage cultures leads to the induction of both class I and class II genes and, as in the case of WEHI-3, this induction does not require the presence of live organisms. These data indicate that several Mycoplasma species have the capacity to induce class II MHC expression in WEHI-3 and both class I and class II MHC expression in bone marrow macrophage cultures in the absence of any T cell products.

Evidence that IFN-gamma does not affect MHC class II gene expression at the post-transcriptional level in a mouse macrophage cell line.

Mouse class II major histocompatibility complex genes have been shown to be regulated at the level of transcription for both tissue-specific and inducible expression. In particular, IFN-gamma induction of the class II genes has been shown to occur at the transcriptional level, although the role that additional post-transcriptional mechanisms of regulation may play in this induction is not known. To evaluate IFN-gamma effects on transcriptional and post-transcriptional events of class II gene expression, we examined the rate of decline of class II transcription, steady-state mRNA, and cell surface protein following the removal of IFN-gamma from maximally stimulated WEHI-3 cells (an IFN-gamma inducible, myelomonocytic cell line). We determined that transcription of class II genes almost completely returned to baseline levels eight hours after removal of IFN-gamma. However, the steady-state level of class II mRNA's required 4 days, and membrane Ia expression required 5 days to return to baseline levels. This decay was linear and allowed us to determine a half-life value of 16-20 h for class II transcripts. These data demonstrate that, following removal of IFN-gamma from fully stimulated cells, transcription of the class II genes declined rapidly, but mRNA was quite long-lived. We also assessed the class II mRNA stability in unstimulated WEHI-3 cells and the B-cell lymphoma. A20/2J, by actinomycin D treatment and northern blot analysis. In agreement with the IFN-gamma washout experiments, transcripts from all four class II genes were quite long-lived in these cell types, with a half-life greater than ten hours. These data support the concept that IFN-gamma acts primarily at the level of class II transcription and argues against IFN-gamma playing a major role in post-transcriptional modulation of class II expression.

Induction of class I and class II MHC antigen expression on murine bone marrow-derived macrophages by IL-4 (B cell stimulatory factor 1).

We have studied the effects of IL-4 (B cell stimulatory factor 1) on the expression of MHC gene products in normal bone marrow-derived macrophages, peritoneal macrophages, and the myelomonocytic cell line WEHI-3. Using both IL-4-containing T cell supernatant and rIL-4, we have observed significant induction of both class I and class II MHC surface expression (about 1.5- to 4-fold increase) in 2-, 3-, and 4-day cultures of bone marrow-derived macrophages. This induction was also apparent at the mRNA level as assessed by Northern blot analysis using A beta, E alpha, and class I probes. Kinetic analysis revealed that induction of class II mRNA by IL-4 was slower than induction by IFN-gamma, requiring 48 h before a significant increase was noted. The magnitude of MHC induction by IL-4 was not as great as that seen with IFN-gamma, which was found to increase surface expression of MHC antigens two- to eightfold. IL-4 also differs from IFN-gamma in the repertoire of macrophages responsive to it. IL-4 was unable to induce class I or class II expression in either thioglycolate-elicited peritoneal macrophages or WEHI-3 cells whereas IFN-gamma induced MHC antigen expression on both cell types under the same conditions. These data demonstrate that IL-4 is capable of inducing both class I and class II MHC gene products in some, but not all, macrophages.