Effect of diesel exhaust particulate (DEP) on immune responses: contributions of particulate versus organic soluble components.

The effect of diesel exhaust particulate (DEP) exposure on innate, cellular and humoral pulmonary immunity was studied using high-dose, acute-exposure rat, mouse, and cell culture models. DEP consists of a complex mixture of petrochemical-derived organics adsorbed onto elemental carbon particles. DEP is a major component of particulate urban air pollution and a health concern in both urban and occupational environments. The alveolar macrophage is considered a key cellular component in pulmonary innate immunity. DEP and DEP organic extracts have been found to suppress alveolar macrophage function as demonstrated by reduced production of cytokines (interleukin-1 [IL-1], tumor necrosis factor- alpha [TNF- alpha]) and reactive oxygen species (ROS) in response to a variety of agents, including lipopolysaccharide (LPS), interferon- gamma (IFN- gamma), and bacteria. Fractionation of DEP organic extract suggests that this activity was predominately in polyaromatic-containing and more polar (resin) fractions. Organic-stripped DEP did not alter these innate pulmonary immune responses. DEP also depressed pulmonary clearance of Listeria monocytogenes and Bacillus Calmette-Guerin (BCG). The contribution of the organic component of DEP is less well defined with respect to acquired and humoral immunity. Indeed, both DEP and carbon black enhanced humoral immune responses (specific immunoglobulin [Ig] E and IgG) in an ovalbumin-sensitized rat model. It is concluded that both the particulate and adsorbed organics may contribute to DEP-mediated immune alterations.

Kinetics of changes in lymphocyte sub-populations in mouse lungs after intrapulmonary infection with M. bovis (Bacillus Calmette-Guerin) and identity of cells responsible for IFNgamma responses.

Gamma interferon (IFNgamma) plays a key role in host defense against pulmonary mycobacterial infections. A variety of lymphocyte subsets may participate in producing pulmonary IFNgamma responses, but their relative contributions after mycobacterial infection have not been clearly elucidated. To address this question, C57Bl/6 female mice were infected by intrapulmonary instillation of 2.5 x 104 BCG (Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium were examined at different time points after the infection. BCG load in lungs peaked between 4 and 6 weeks post-infection and declined to very low levels by the 12th week of infection. Recovery of lung interstitial lymphocytes doubled by 4-6 weeks after infection and declined thereafter. Flow cytometric analysis of the lung-derived lymphocytes revealed that about 5% of the these cells made IFNgamma in control mice, and this baseline IFNgamma production involved T (CD3+NK1.1-), NK (CD3-NK1.1+) and NKT (CD3+NK1.1+) cells. As the BCG lung infection peaked, the total number of CD3+ T cells in the lungs increased threefold at 5-6 weeks post-infection. There was a marked increase (sixfold) in the number of T cells secreting IFNgamma 5-6 weeks post-infection. Some increase was also noted in the NKT cells making IFNgamma, but the numbers of NK cells making IFNgamma in BCG-infected lungs remained unaltered. Our results suggest that whereas NK and NKT cells contribute to baseline IFNgamma secretion in control lungs, expansion in the IFNgamma-producing T-cell population was essentially responsible for the augmented response seen in lungs of BCG-infected mice.

Antimalarial effect of cyclosporin-A on murine P. berghei and human P. falciparum.

The effects of Cyclosporin-A (CsA) on the growth of Plasmodia were investigated in an experimental murine model in vivo and on human malaria in vitro. Mice were inoculated with Plasmodium berghei and then treated with different doses of CsA at the patent period. The development and course of this normally lethal parasitaemia in mice was affected by treatment with CsA which is a known immunosuppressant. The drug showed complete protection at a dose of 20 mg/kg wt/day without any recrudescence. Antibody level was at the detection limit after first bout of drug-cured infection. CsA was found to be an inhibitor of P. falciparum growth in a dose dependent fashion, as the concentrations of drug in culture medium increased, a significant reduction in parasitaemia was observed.

Natural killer activity against human K562 tumour cells during Plasmodium cynomolgi malarial infection of rhesus monkeys.

This study assessed the natural killer (NK) cell activity profile during Plasmodium cynomolgi infection in rhesus monkeys. There was a significant decrease in the NK cell activity in the peripheral blood leukocytes of infected monkeys during the early, ascending phase of infection. However, as the parasite load decreased, NK cell activity returned to normal levels. This could be correlated with the peak increase in lymphocyte counts. This indicated that a decrease in NK cell activity observed at an earlier stage during an active P. vivax malarial infection was a temporary phenomenon.

Effect of cyclosporin-A, antimalarial drug, on lymphocyte proliferation of Balb/c mice in vitro.

Cyclosporin-A(CsA) caused inhibition of lymphocyte proliferation at higher doses (5 and 10 micrograms/ml) compared to controls. When spleen cells were preincubated with high doses of CsA and washed, the normal lymphocyte response to stimulation with mitogen (concanavalin-A) and lymphokine (interleukin-2) was not affected. The results indicate that CsA's suppressive effect at higher doses, was a temporary one and potential use of CsA to control parasitic infections should be examined.

The natural occurrence of circulating antibodies in populations of endemic malarious areas.

The magnitude of humoral response to soluble antigen extracted from Plasmodium falciparum schizonts and merozoites was assessed in 744 blood samples collected from different parts of India. In this study, parasitological and immunological data were considered for assessment of antibody during natural infections at various seasons. The antibody response has been measured by enzyme-immuno assay. Survey was done in all age groups. Overall antimalarial IgG level had started increasing after five years which shows that the rate of infection was high in the small age groups. Elevated level of IgG in populations indicates that the study area is undergoing a period prevalence of the disease. In most of the individuals with active infection, IgG and IgM levels were high. Positivity in IgM denoted the active transmission of malaria. Elevated levels of antigen specific IgA was observed in some cases but the mechanism is not yet understood. Presence of circulating immune complexes during acute infection shows the failure of detection of circulating free antibodies in some individuals. The significance of findings in relation to serological status in individuals exposed naturally to malaria has been discussed.

Properties and characterization of a rat spleen cell-derived factor that induces resistance to natural killer cell lysis in YAC lymphoma cells.

Supernatants of Con A-stimulated rat spleen cell cultures contain a factor that induces relative resistance to NK cell-mediated cytotoxicity in the YAC cell line, a line that is otherwise highly susceptible to murine NK cell-mediated lysis. This NK-lysis resistance-inducing factor (LRIF) has a Mr of 12,600 Da, as determined by gel filtration chromatography, and an isoelectric pH of 4.8. NK-LRIF is heat labile and is de-activated by treatment with proteolytic enzymes. Unlike immune-IFN (IFN-gamma), NK-LRIF is not inactivated by pH 2 treatment, and antibodies capable of neutralizing IFN-alpha and IFN-gamma do not abrogate the effect of NK-LRIF. Highly purified IL-2 preparations lack NK-LRIF activity. NK-LRIF does not induce a general resistance to lysis in YAC cells, because control and NK-LRIF-treated YAC cells were equally susceptible to alloimmune cytotoxic T cells. YAC cells treated with NK-LRIF showed a marked enhancement (5- to 10-fold) in the expression of class I MHC Ag. This observation supports the proposition that the NK susceptibility of target cells could be inversely related to the expression of class I MHC Ag.

Lectin-induced cytotoxic activity in spleen cells from young and old mice. Age-related changes in types of effector cells, lymphokine production and response.

Concanavalin A (Con A)-induced cytotoxic activity, interferon (IFN) and interleukin-2 (IL-2) levels in cultures of spleen cells from young (2-3 months) and old (22-24 months) C57BL/6 female mice were studied. Con A-activated spleen cells from old mice attained significantly higher cytotoxic activity compared with activated spleen cells from young mice. Activated spleen cells from old and young mice showed differences in their ability to lyse different types of target cells. Both could lyse P-815 cells, neither could lyse K562 cells, and only activated cells from old mice could lyse EL-4 cells. Cytotoxic spleen cells from the old mice were more sensitive to anti-asialo-GM-1 and anti-Lyt-2.2 plus complement (C) treatment. While levels of IL-2 produced by spleen cells from young mice were higher, the addition of exogenous IL-2 had no effect on cytotoxic activity of the spleen cells from old mice. Exogenous IL-2, however, could lower cytotoxic activity of Con A-activated spleen cells from young mice. Activated spleen cells from old mice generated higher levels of IFN-gamma while the addition of an anti-IFN-gamma antibody boosted the level of cytotoxicity by Con A-activated spleen cells from young mice. These results suggest that IFN-gamma may act as a feedback inhibitory signal regulating the levels of cytotoxicity induced in spleen cells from young mice in response to Con A. The cytotoxic activity generated in Con A-activated spleen cells from old mice reflects a defect in this feed-back regulation.

Effect of feeding a diet with half of the recommended levels of all vitamins on the natural and inducible levels of cytotoxic activity in mouse spleen cells.

Groups of 6-week-old female C57Bl/6 mice were fed a normal diet with recommended levels of all vitamins or a vitamin-deficient (VD) diet containing half of the recommended level of each vitamin. At different time periods (1-11 weeks) after the initiation of diets, basal natural killer (NK) activity, interleukin-2 (IL-2) and concanavalin A (Con A)-induced cytotoxic activity, Con A-induced IL-2 production and levels of allospecific cytotoxic T cell activity generated in a mixed lymphocyte culture (MLC), were studied in spleen cells derived from control and VD mice. Results indicated that: (i) spleen NK activity remained normal until 2 weeks after the initiation of VD diet, fell steeply to low levels at the 4 and 5 week time points and remained depressed thereafter; (ii) IL-2- and Con A-induced levels of cytotoxic activity in spleen cells derived from VD mice declined at 4 weeks after the institution of VD diet, and then remained low throughout the study; (iii) the capacity of spleen cells from VD mice to generate IL-2 in response to Con A and cytotoxic T cells in response to allogeneic spleen cells, was normal at 1 and 4 weeks after initiation of the VD diet and was markedly depressed at the 6 and 9 week time points. These results suggest that partial combined deficiencies of dietary vitamins strongly influence assays of immune function.

Interleukin-2-induced activation of natural killer activity in spleen cells from old and young mice.

Generation of natural killer (NK) activity in response to a partially purified preparation of rat interleukin-2 (IL-2) was compared in spleen cells derived from young (8-10 weeks old) and old (greater than 2 years old) female C57BL/6 mice. Significant NK activation was observed in both young and old mouse spleen cells incubated with 100 U IL-2/ml for 1-4 days, but the levels of cytotoxic activity generated in old mouse spleen cells was always lower than those of similarly treated young mouse spleen cells. Differences in IL-2-induced NK activation in old and young mouse spleen cells was obtained irrespective of the concentration of IL-2 used (25-400 U/ml). Quantitative comparisons indicated that old spleen cells activated by 3 day incubation with IL-2 acquired about two-fold higher NK activity than fresh young mouse spleen cells but still had only one-fourth of the levels of NK activity attained by IL-2-activated young mouse spleen cells. Cytotoxic activity of IL-2-activated young or old mouse spleen cells were totally abrogated by anti-asialo GM-1 antiserum + C but not by anti-Ly-2 + C treatment, indicating that the activated cytotoxic cells fell in the NK cell category. An analysis of NK precursor (NK-p) frequency by limiting dilution assay indicated that the NK-p frequency was about 4-fold higher in young as compared to old mouse spleen cells. The level of cytotoxic activity attained per NK-p cell was not significantly different for NK-p cells of old or young mice.

Effect of protein calorie malnutrition on the levels of natural and inducible cytotoxic activities in mouse spleen cells.

Six-week-old C57B1/6 female mice were fed a normal (24% protein) or an isocaloric but protein-deficient (4% protein) diet. At different time periods after the initiation of diets, basal natural killer (NK) activity, interleukin-2 (IL-2) and concanavalin-A (Con-A)-induced cytotoxic activity, Con-A-induced IL-2 production and levels of allospecific cytotoxic T cell activity generated in a mixed lymphocyte culture (MLC), were studied in spleen cells derived from control and protein deficient (PD) mice. Results indicated that (a) levels of spleen NK activity increased initially in PD mice, but after 7 weeks on PD diet declined to normal and subnormal levels, (b) IL-2 generation in response to Con-A as well as IL-2 activation of NK activity were comparable in spleen cells of control and PD mice at all time points tested, (c) Con-A-induced cytotoxic activity was significantly greater in spleen cells from PD mice, the difference being greater at higher doses of Con-A, and (d) generation of alloimmune cytotoxic T cells in a MLC reaction was normal in PD mouse spleen cells until 4 weeks after the beginning of PD diet, but declined markedly thereafter. Relevance of these observations to other related findings in protein calorie malnutrition are discussed.

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice.

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.

Identity of effector cells participating in the reverse antibody-dependent cell-mediated cytotoxicity.

Our previous work has shown that antibody-coated mouse spleen cells express enhanced cytotoxic activity against some Fc-receptor-bearing target tumour cells by a mechanism which appears to be similar to an antibody-dependent cell-mediated cytotoxicity (ADCC) reaction with reversed polarity of the antibody bridge (R-ADCC). In this report we have shown that (i) the levels of basal natural killer (NK), ADCC and R-ADCC cytotoxic activities in mouse spleen cells are strongly correlated with each other, (b) simultaneous induction of ADCC and R-ADCC reactions does not result in an additive cytotoxic response, and (iii) YAC cells which do not bear Fc receptors and are highly sensitive to lysis by NK cells, can specifically and competitively inhibit the ADCC and R-ADCC reactions. These results suggest that the R-ADCC reaction may be mediated by the same effector cell population as mediates NK and ADCC reactions against tumour target cells.

Regulation of natural killer activity in vivo. III. Effect of hypophysectomy and growth hormone treatment on the natural killer activity of the mouse spleen cell population.

Spleen natural killer (NK) activity against YAC tumor cells was reduced in female C57BL/6J mice hypophysectomized at 3 weeks and sacrificed 4-8 weeks later. Proportions of null cells and of target binding cells in spleens from hypophysectomized and sham operated mice were significantly different. Administration of ovine growth hormone (GH) (100 microgram/day, i.p. for 10 days) resulted in a marked recovery of NK activity in hypophysectomized mice. NK activity by spleen cells from hypophysectomized or sham operated mice (with or without GH treatment) was mediated by non-T, non-B lymphocytes.