Tuberculosis and risk of acute myocardial infarction: a propensity score-matched analysis.

Several pathogens have been associated with increased cardiovascular disease (CVD) risk. Whether this occurs with Mycobacterium tuberculosis infection is unclear. We assessed if tuberculosis disease increased the risk of acute myocardial infarction (AMI). We identified patients with tuberculosis index claims from a large de-identified database of ~15 million adults enrolled in a U.S. commercial insurance policy between 2008 and 2010. Tuberculosis patients were 1:1 matched to patients without tuberculosis claims using propensity scores. We compared the occurrence of index AMI claims between the tuberculosis and non-tuberculosis cohorts using Kaplan-Meier curves and Cox Proportional Hazard models. Data on 2026 patients with tuberculosis and 2026 propensity-matched patients without tuberculosis were included. AMI was more frequent in the tuberculosis cohort compared with the non-tuberculosis cohort, 67 (3·3%) vs. 32 (1·6%) AMI cases, respectively, P < 0·01. Tuberculosis was associated with an increased risk of AMI (adjusted hazard ratio (HR) 1·98, 95% confidence intervals (CI) 1·3-3·0). The results were similar when the analysis was restricted to pulmonary tuberculosis (adjusted HR 2·43, 95% CI 1·5-4·1). Tuberculosis was associated with an increased risk of AMI. CVD risk assessment should be considered in tuberculosis patients. Mechanistic studies of tuberculosis and CVD are warranted.

Increased weight loss with reduced viral replication in interleukin-10 knock-out mice infected with murine cytomegalovirus.

The anti-inflammatory cytokine interleukin (IL)-10 plays an important role in the regulation of host-immune responses. Here we studied the role IL-10 plays in host responses to cytomegalovirus (CMV) infection. We demonstrate that manifestations of murine CMV (MCMV) disease are more severe in IL-10 knock-out mice, despite significantly reduced levels of viral replication. Cytokine analysis of serum revealed increased levels of interferon (IFN)-gamma, monocyte chemotactic protein 1 (MCP-1) and IL-6, all of which are potent stimulators of inflammatory responses. Depletion of IFN-gamma by monoclonal antibodies in IL-10 knock-out mice failed to improve the physical condition of the mice, while increasing viral replication. In contrast, serum levels of IL-6 in the knock-out animals were unaffected by IFN-gamma depletion and remained significantly elevated early in the course of infection. These data suggest that increased weight loss observed in IL-10 knock-out mice may be attributed to the uncontrolled production of proinflammatory cytokines, including IL-6.

Effects of antiretroviral therapy on immunity in patients infected with HIV.

Drug therapy for human immunodeficiency virus (HIV) is highly effective in suppressing viral replication and restoring immune function in patients with HIV. However, this same treatment can also be associated with immunotoxicity. For example, zidovudine and various other antiretroviral agents are capable of causing bone marrow suppression. Agents used to treat opportunistic infections in these individuals, including ganciclovir, foscarnet, and sulfamethoxazole-trimethoprim, can cause additional hematotoxicity. Drug-drug interactions must also be considered and managed in order to control iatrogenic causes of immunotoxicity. In this review, we examine the normal immune response to HIV, and the benefits of antiretroviral therapy in prolonging immune function. We then discuss immune-related adverse effects of drugs used to treat HIV and the opportunistic infections that are common among these patients. Finally, we address in vitro, animal, and clinical evidence of toxicity associated with various combination use of these agents.

Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo.

Cyclic ADP-ribose is believed to be an important calcium-mobilizing second messenger in invertebrate, mammalian and plant cells. CD38, the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo. Using CD38-deficient mice, we demonstrate that the loss of CD38 renders mice susceptible to bacterial infections due to an inability of CD38-deficient neutrophils to directionally migrate to the site of infection. Furthermore, we show that cyclic ADP-ribose can directly induce intracellular Ca++ release in neutrophils and is required for sustained extracellular Ca++ influx in neutrophils that have been stimulated by the bacterial chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP). Finally, we demonstrate that neutrophil chemotaxis to fMLP is dependent on Ca++ mobilization mediated by cyclic ADP-ribose. Thus, CD38 controls neutrophil chemotaxis to bacterial chemoattractants through its production of cyclic ADP-ribose, and acts as a critical regulator of inflammation and innate immune responses.

Neonatal T cells in an adult lung environment are competent to resolve Pneumocystis carinii pneumonia.

Initiation of the pulmonary inflammatory response to Pneumocystis carinii is delayed by 3 wk in mice infected as neonates compared with adults. There was no difference in the proliferative response of draining lymph node T cells from mice infected as neonates compared with adults when stimulated in vitro with either Con A or anti-CD3 mAB: However, TNF-alpha and IFN-gamma mRNA expression in the lungs of P. carinii-infected neonates was significantly lower than in adults indicating a lack of appropriate activation signaling in the local environment. This may have been due to active suppression because TGF-beta mRNA expression was significantly elevated in neonatal lungs compared with adults. To determine whether T cells from 10-day-old mice would effect resolution of P. carinii if harbored in an adult lung environment, cells were adoptively transferred to SCID mice with established P. carinii infections. There was no difference in the kinetics of T cell migration into the lungs or of clearance of P. carinii organisms when SCID mice were reconstituted with splenocytes from young mice as compared with adult mice. Furthermore, splenocytes from young mice stimulated both TNF-alpha and IFN-gamma mRNA expression to levels that were similar to that in the lungs of SCID mice reconstituted with adult cells. These data indicate that neonatal lymphocytes are competent to resolve P. carinii infection when harbored in an adult lung environment, suggesting that the neonatal lung environment, and not the T cells, is ineffective at responding to P. carinii infection.

Delayed inflammatory response to Pneumocystis carinii infection in neonatal mice is due to an inadequate lung environment.

Challenge of neonatal mice with an intranasal inoculation of Pneumocystis carinii results in a subclinical infection that takes 6 wk to resolve, whereas adult mice resolve a comparable challenge within 3 wk. This delayed clearance is due to a delayed inflammatory response in neonatal mice; however, the reason for this delay has been unknown. To determine whether the neonatal lung environment is sufficient to attract immunocompetent lymphocytes into the lungs, an adoptive transfer strategy was employed in which splenocytes from adult BALB/c mice were transferred into P. carinii-infected neonatal or adult SCID mice. All adults, but no pups, resolved their infections by day 37 postreconstitution. Despite reconstitution with adult splenocytes, pups had a negligible lung inflammatory response until day 24, whereas adult mice had activated CD4(+) and CD8(+) cells in the lung by day 13. The delay in neonates corresponded to delayed kinetics of expression of lung cytokines TNF-alpha and IFN-gamma mRNA and chemokines lymphotactin, RANTES, and macrophage inflammatory protein-1ss mRNA. Phagocytic cells from neonatal mice were significantly less efficient than adult cells at migrating to the draining lymph nodes after phagocytosing fluorescent beads. There were fewer dendritic cells and Ia(+) myeloid cells in the lungs of P. carinii-infected neonatal mice compared with adults. These data indicate that the lung environment of neonatal mice is insufficient for migration of T cells, due at least in part to inefficient phagocytosis and migration of APCs to the lymph nodes as well as delayed chemokine and TNF-alpha mRNA expression.

Recognition of Pneumocystis carinii antigens by local antibody-secreting cells following resolution of P. carinii pneumonia in mice.

To examine the repertoire of Pneumocystis carinii antigens recognized by antibody-secreting B cells from tracheobronchial lymph nodes isolated immediately following recovery from P. carinii pneumonia, monoclonal antibodies (MAbs) were produced from these cells. In contrast to previous studies of systemic immunity, P. carinii gpA was not the immunodominant antigen recognized by these B cells. Forty-nine (91%) of 54 P. carinii-specific hybridoma culture supernatants reacted with P. carinii antigens other than gpA. Many of the resulting MAbs recognized a previously uncharacterized antigen expressed on the surface of both cysts and trophozoites. Western blotting using one of the cloned MAbs revealed reactivity with a broad range of antigenic material, with the most intense reactivity in the 50- to 65-kDa region of the blot. The antigens identified by these MAbs merit further investigation regarding protective immunity to P. carinii because they were recognized by B cells in the context of recovery from P. carinii pneumonia.

Protection against Pneumocystis carinii pneumonia by antibodies generated from either T helper 1 or T helper 2 responses.

To determine whether different antibody isotypes associated with T helper 1 (Th1) or Th2 responses are protective against Pneumocystis carinii, mice with disrupted interleukin 4 genes (IL-4(-/-) mice) or gamma interferon genes (IFN-gamma(-/-) mice) along with wild-type C57BL/6 mice were immunized intratracheally against P. carinii, depleted of T cells in vivo by use of monoclonal antibodies, and rechallenged intratracheally with 10(7) viable P. carinii organisms. Nearly all immunized mice resolved their lung P. carinii infections (limit of detection, log10 4.06) within 21 days of challenge even though they were depleted of T cells. Unimmunized mice depleted of T cells had significant lung infections (>log10 5.5) at day 21 post-P. carinii challenge. IFN-gamma(-/-) and wild-type mice developed P. carinii-specific immunoglobulin primarily of the immunoglobulin G1 (IgG1) subclass with relatively little P. carinii-specific IgG2a, IgG2b, or IgG3 in their sera, characteristic of a Th2-type response. In contrast, IL-4(-/-) mice had primarily an IgG2b P. carinii-specific antibody response in their sera with very little IgG1. Although IgG2b was the predominant isotype in IL-4(-/-) mice, optical density values of IgG2a and IgG3 were significantly higher in these mice (two and three times, respectively) than in IFN-gamma(-/-) mice, characteristic of a Th1-type response. Together, these data indicate that resolution of P. carinii infection can be mediated by specific antibody responses and that the antibody response can be either a predominantly Th1 or Th2 type. Furthermore, although wild-type mice mounted a Th2-like antibody response, IL-4(-/-) mice could resolve P. carinii pneumonia, indicating that resistance to P. carinii can occur in the absence of IL-4.

Acquired resistance to Cryptococcus neoformans in adult mice vaccinated as newborns.

Although Cryptococcus neoformans causes serious infections in AIDS patients, cryptococcosis in immunologically immature infants, as in immunocompetent adults, is rare. To investigate the resistance of neonates to C. neoformans and to determine whether they could be efficiently vaccinated as neonates against challenge with the yeast as adults, the course of infection was monitored in the lungs of mice infected intranasally with yeast cells. Neonates were less able than adults to reduce yeast burdens less than 24 h postinoculation and less able to control the progressive growth of yeast over several weeks. However, in both neonates and adults, yeasts were substantially eradicated by 6 to 8 weeks after infection. Numbers of all classes of leukocytes recovered from lung lavages of infected neonates and adults were similar. Significant differences appeared only on day 14, when neonates had more neutrophils and adults had significantly more CD4+ CD45RB cells with low fluorescence intensity. When vaccinated neonates were rechallenged after reaching adulthood, they expressed resistance to C. neoformans as effectively as did mice immunized as adults and survived an intravenous challenge that was lethal for unimmunized controls. Thus, exposure of neonatal mice to viable C. neoformans yeast, which persists in the lungs for many weeks, does not result in immunological tolerance to a yeast challenge in adult mice, as predicted by immunological dogma, but instead immunizes them. Therefore, even in immunologically immature individuals, the immune system serves to protect against pathogens rather than simply to distinguish self from nonself.

Neutralization of interferon-gamma exacerbates pneumocystis-driven interstitial pneumonitis after bone marrow transplantation in mice.

The role of IFNgamma in the development of infection-driven interstitial pneumonitis in a model of murine graft-versus-host disease was investigated. Mice were given either syngeneic or allogeneic bone marrow transplants along with lung Pneumocystis carinii infections and were treated with either control mAb or anti-IFNgamma mAb. At day 21 after transplant, lung weights were elevated nearly twofold in all groups. By day 41, mice in all groups had cleared the P. carinii but only the mice given allogeneic transplants and anti-IFNgamma had increased lung weights. Increased lung weights in the anti-IFNgamma-treated mice corresponded to alveolar infiltration of eosinophils, neutrophils, and multinucleated giant cells and exacerbated interstitial pneumonitis compared with mice treated with control antibody. Intracellular staining indicated that there were 3- to 10-fold more CD4+ cells producing IFNgamma than those producing IL-4 in the lung lavages of mice given either syngeneic or allogeneic transplant. Treatment of transplanted mice with anti-IFNgamma resulted in a significant decrease in IFN-gamma-producing CD4+ and CD8+ cells in the lung lavages but no change in the number of IL-4-producing CD4+ cells. These data indicate that IFNgamma is critical for controlling the development of P. carinii-driven interstitial pneumonia after either syngeneic or allogeneic bone marrow transplant in mice.

Role of gamma interferon in the host immune and inflammatory responses to Pneumocystis carinii infection.

The role of gamma interferon (IFN-gamma) in host defense to Pneumocystis carinii was investigated by use of three different murine models of infection. C57BL/6 scid/scid (severe combined immunodeficient [SCID]) mice were given intratracheal inoculations of P. carinii and reconstituted with splenocytes from either mice with disrupted IFN-gamma genes (IFN-gamma-/- mice) or homozygous wild-type (IFN-gamma+/+) mice. Unreconstituted SCID mice had log10 7.08 +/- 0.13 P. carinii nuclei in their lungs at day 22 postinfection, whereas SCID mice reconstituted with splenocytes from either wild-type or IFN-gamma-/- mice had cleared the infection. However, there was a prolonged and exacerbated inflammatory response in the lungs of SCID mice reconstituted with IFN-gamma-/- splenocytes which was characterized by interstitial pneumonia, eosinophilia, and multinucleated giant cell formation. Similar results were found in C.B17 SCID mice reconstituted with CD4+ cells from P. carinii-immunized donors treated with neutralizing anti-IFN-gamma monoclonal antibody (MAb). These mice resolved their P. carinii infections; however, they also exhibited exacerbated lung pathology compared with mice treated with a control MAb. Finally, IFN-gamma-/- mice challenged intratracheally with P. carinii resolved their infection within 56 days as did IFN-gamma+/- mice. Furthermore, depletion of T cells in vivo with a MAb resulted in IFN-gamma-/- mice becoming susceptible to P. carinii infection. Together, these data indicate that IFN-gamma is not required for resolution of P. carinii infection; however, in the absence of IFN-gamma, there is a prolonged and exacerbated P. carinii-driven interstitial pneumonia characterized by eosinophilia and formation of multinucleated giant cells.

Susceptibility to Pneumocystis carinii infection: host responses of neonatal mice from immune or naive mothers and of immune or naive adults.

Mice from either naive or immunized dams were given intranasal inoculations of Pneumocystis carinii as neonates (24 to 48 h old). Lung P. carinii burdens increased through day 13 postinoculation in all pups and declined to nearly undetectable numbers by day 23 in pups from immune mothers. However, P. carinii numbers in pups from naive mothers did not begin to decline significantly until after day 33, and P. carinii organisms were still detectable in low numbers through day 45. In contrast, the lungs of naive or immunized adult mice contained detectable numbers of P. carinii organisms only up to 9 or 3 days, respectively, after inoculation. The onset of clearance of P. carinii organisms from the lungs of neonatal mice and naive adults was coincident with infiltration of neutrophils and CD4+ CD45RBlo cells into the alveolar spaces and increased titers of P. carinii-specific antibody in sera. Immunized dams had high levels of P. carinii-specific antibody in both their sera and milk, and pups from these dams had higher titers of P. carinii-specific antibody than did pups from naive dams. These data indicate that P. carinii survives for a much longer period in neonates than in adult mice, which is the result of a delay in the onset of the immune response in neonates. Furthermore, immunized mothers contributed to an early clearance of P. carinii organisms by their offspring presumably because of the transfer of P. carinii-specific antibody. However, the passively acquired antibody did not seem to have an effect until the neonates began to mount their own responses.

The importance of neutrophils in resistance to pneumococcal pneumonia in adult and neonatal mice.

Neonatal mice succumbed to intranasally-inoculated Streptococcus pneumoniae doses which were as much as 250 times less than the doses that adult mice were resistant to. Neutrophil migration into lungs of neonates was similar in kinetics and intensity to that in adults in response to lethal doses of S. pneumoniae. Interestingly, neutrophil infiltration into the lung alveoli of neonates occurred at lower doses of bacteria than that required for similar responses in adults. Furthermore, depletion of neutrophils in adult and neonatal mice inoculated with low doses of bacteria resulted in significantly higher lung burdens of bacteria in neonatal mice as compared to adults. These data indicate that increased susceptibility of neonates to S. pneumoniae is not the result of incompletely developed neutrophil function and infact, indicate that neutrophils contribute more to resistance to low doses of S. pneumoniae in neonates than they do in adult mice.

The role of T cells in infection-driven interstitial pneumonia after bone marrow transplantation in mice.

Over the course of five weeks, there were no significant histopathological changes in the lungs of mice given either allogeneic or syngeneic bone marrow transplants (BMT) with whole body irradiation (WBI). However, all mice that received both WBI and Pneumocystis carinii inoculation developed a more protracted and severe interstitial pneumonia than that of normal mice given P carinii. This pneumonia was exacerbated by allogeneic BMT but was ameliorated by syngeneic BMT. The interstitial pneumonia caused by allogeneic BMT and P carinii infection was associated with the influx of large numbers of activated CD4+ cells of donor origin. Depletion of CD4+ T cells in vivo in these mice inhibited both the development of graft-versus-host disease (GVHD) and the interstitial pneumonia. In vitro depletion of T cells before allogeneic BMT and P carinii infection also inhibited GVHD-however, it did not stop the development of interstitial pneumonia caused by the infiltration of host-derived T cells. These results indicate that in this model infection is required for development of interstitial pneumonia after allogeneic BMT. This interstitial pneumonia can be caused by the accumulation of CD4+ cells of either donor or recipient origin but not by CD8+ cells. The accumulation of these cells in lungs of normal mice in response to P carinii does not cause interstitial pneumonia, but irradiation of the host before the cellular accumulation does. This interstitial pneumonia can occur in infected mice after either syngeneic or allogeneic BMT, but is exacerbated by GVHD.

Antibody-mediated shift in the profile of glycoprotein A phenotypes observed in a mouse model of Pneumocystis carinii pneumonia.

It is well established that Pneumocystis carinii has the molecular capability for variation of a major surface antigen, glycoprotein A (gpA). However, the extent of expression of gpA variation among P. carinii organisms infecting a single host and whether this variation has any impact on host-parasite immunological interactions is unknown. Using a mouse model of P. carinii pneumonia, we were able to demonstrate the expression of more than one gpA phenotype in a closed population of infected mice. Administration of monoclonal antibody (MAb) 2B5, which is specific for one of the gpA phenotypes, resulted in a marked diminution in the frequency of this particular gpA phenotype in the population of organisms. This effect was due to a loss of trophozoites bearing the specific epitope recognized by MAb 2B5; cysts bearing the same epitope appeared unaffected. Interestingly, P. carinii was unable to introduce a new phenotype into the population to compensate for the loss of trophozoites bearing the epitope recognized by MAb 2B5. Discontinuing administration of MAb 2B5 allowed the MAb 2B5-binding phenotype to reemerge. This finding suggests that the phenotype recognized by MAb 2B5 was continually produced even when MAb 2B5 was present. Thus, although P. carinii exhibited a form of antigenic variation, it did not appear able to rapidly introduce new phenotypes into the population in response to destruction by antibodies.

Autoantibodies inhibit interleukin-7-mediated proliferation and are associated with the age-dependent loss of pre-B cells in autoimmune New Zealand Black Mice.

Surface IgM+B220+ B cell precursors can be categorized as either leukosialin (CD43/S7) negative (late stage pre-B cells) or positive (pro-B/early pre-B cells). In autoimmune New Zealand Black (NZB) mice, bone marrow small pre-B cells (IgM-CD43-B220+) and pro-B/early pre-B cells (IgM-CD43+B220+) declined significantly with age. In particular, subpopulations of pro-B/early pre-B cells expressing the heat stable antigen (HSA) were found in lower proportions with age. Significant decreases in interleukin-7 (IL-7) colony forming units (CFU) were also seen in NZB mice by 6 to 8 months of age and accompanied alterations in the numbers of pro-B and pre-B cells in bone marrow. Concomitant with reduced numbers of B lineage precursor cells and IL-7 CFU in vivo, NZB mice produced serum IgM antibodies that strongly inhibited IL-7 CFU responses in vitro. Two monoclonal IgM antibodies (5G9, 2F5) derived from LPS stimulated 10-month-old NZB splenocytes recognized pre-B cell surface antigens on both pre-B cell lines and on IL-7 stimulated bone marrow pro-B/pre-B cells. However, these monoclonal antibodies (MoAb) failed to significantly stain ex vivo bone marrow cells. The 5G9 and 2F5 MoAbs also partially inhibited IL-7 CFU in vitro. These results suggest that NZB bone marrow becomes increasingly deficient in B cell precursors and especially in IL-7 responsive pre-B cells with age. IgM serum antibodies and monoclonal IgM antibodies derived from older NZB mice inhibit pre-B cell growth to IL-7. The production of such autoantibodies may interfere with B cell development in aging NZB mice by preventing IL-7-mediated proliferation.

B220- bone marrow progenitor cells from New Zealand black autoimmune mice exhibit an age-associated decline in Pre-B and B-cell generation.

New Zealand Black (NZB) autoimmune mice exhibit progressive, age-dependent reduction in bone marrow pre-B cells. To ascertain the capacity of NZB bone marrow B220- cells to generate pre-B cells in a supportive environment, B-lineage (B220+) cell-depleted and T-cell-depleted bone marrow cells from NZB mice at 1 to 3, 6, and 10 to 11 months of age were adoptively transferred into irradiated (200R) C.B17 severe combined immunodeficient (SCID) mice. Bone marrow pre-B cells (sIgM- CD43[S7]- B220+) were assessed 3 and 10 weeks posttransfer. Pre-B cells and B cells were reconstituted in SCID recipients of older NZB progenitor cells by 10 weeks posttransplant, in contrast to the very low numbers of pre-B cells present in the donor bone marrow. However, B220- bone marrow progenitor cells from greater than 10-month-old NZB donors were deficient in the reconstitution of both pre-B and B cells in SCID recipients at 3 weeks post-transfer. This reflected a slower kinetics of repopulation, because older NZB-->SCID recipients had numbers of both pre-B and B cells similar to recipients of young NZB progenitor cells by 10 weeks posttransplant. Adoptive transfer of equal mixtures of BALB/c and older NZB bone marrow B220- progenitor cells into irradiated C.B17 SCID recipients failed to demonstrate active suppression. These results suggest that, with age, NZB bone marrow has reduced numbers and/or function of early B220- B-lineage progenitors. Consistent with this hypothesis, B220- bone marrow cells from older NZB mice were deficient in progenitors capable of yielding interleukin-7 (IL-7) responsive pre-B cells in vitro on stimulation with the pre-B-cell potentiating factor, insulin-like growth factor 1 (IGF-1).

Apoptosis accompanies a change in the phenotypic distribution and functional capacity of murine bone marrow B-cells chronically exposed to prednisolone.

Prednisolone (PD) is commonly used for the treatment of inflammation accompanying diseases such as arthritis, allergy, asthma, and autoimmunity. While it is well documented that PD induces apoptosis in immature T-cells of the mouse, the effects of PD on development of immature B-cells in normal bone marrow (BM) was not known. An implantation system was developed which chronically delivered PD at a rate of a few nanograms per milliliter of plasma to mice. Ten days of exposure to such levels of PD caused splenic and thymic atrophy, which was accompanied by a 50% decrease in the numbers of circulating lymphocytes. Flow cytometric analysis (FACS) of the effects of PD on the BM revealed a threefold decrease in the proportion of B220+IgM- pre-B-cells and immature IgM+IgD- B-cells. However, the mature IgM+IgD+ cells were reasonably resistant to the effects of PD. A 25% decrease in small nucleated cells presumed to be part of the lymphocyte compartment was also noted from the scatter profiles of the marrow of PD-treated mice. These marked changes in BM composition were also accompanied by significant reductions in capacity of the BM to respond to trinitrophenylated-lipopolysaccharide (TNP-LPS) after exposure to PD either in vivo or in vitro. Studies to ascertain whether apoptosis played a role in the decline in the number of developing B-cells of marrow exposed to PD were performed in vitro in order to reduce the possibility of phagocytosis of apoptotic cells. A recent modification of FACS cell cycle analysis, which is highly quantitative and allows rapid analysis of heterogeneous tissues such as the marrow, was used to detect the apoptotic cells. After 16 hr of culture in 10(-7) M PD, approximately 40% of IgM+ and B220+ cells of BM resided to the left of G0/G1 in a region associated with apoptotic cells previously termed the A0 or "hypodiploid" region. Thus, these data indicate that chronic exposure to low levels of PD significantly altered the B-cell compartment of the murine bone marrow both in vivo and in vitro, potentially inducing apoptosis in these cells.

IFN-gamma abrogates IL-7-dependent proliferation in pre-B cells, coinciding with onset of apoptosis.

These studies investigated the mechanism by which interferon-gamma (IFN-gamma) inhibits the interleukin-7 (IL-7)-dependent proliferation of BALB/c bone marrow B-cell precursors in vitro. Low concentrations (1 U/ml) of recombinant murine IFN-gamma (rmIFN-gamma) caused a approximately 80% suppression of IL-7 colony-forming units (CFU) formation in semi-solid media, in part through a direct affect on isolated B220+ pre-B cells. IFN-gamma did not induce apoptosis in small resting pre-B cells in BALB/c bone marrow. There was no difference in the proportion of apoptotic B220+ pre-B cells in IFN-gamma-treated cultures compared to cultures treated with IL-7 alone. However, IL-7-responsive pre-B cells generated from bone marrow had a 30-50% loss in cells in S+G2/M phases of the cell cycle and an increase of up to twice as many in apoptotic cells within 48 hr of exposure to IFN-gamma. Notably, expression of the tyrosine phosphatase B220 was increased in the IFN-gamma-treated pre-B cells. Interestingly, although there was no substantial change in IL-7 receptor mRNA expression upon IFN-gamma treatment, a small decrease in binding of biotinylated IL-7 to IFN-gamma-treated pre-B cells was observed. These results suggest that IFN-gamma inhibits IL-7 responsiveness in pre-B cells, resulting in a subtle down-regulation of IL-7 binding, inhibition of proliferation and, ultimately, apoptosis.