Production of interleukin-8, RANTES and MCP-1 in intrinsic and extrinsic asthmatics.

The aim of this study was to determine the relative production of chemokines interleukin-8 (IL-8), regulated on activation, normal T-cell expressed and secreted (RANTES) and monocyte chemotactic protein-1 (MCP-1) by intrinsic and extrinsic asthmatics. Nine intrinsic asthmatics, 10 extrinsic asthmatics, five nonatopic and five atopic controls underwent bronchoalveolar lavage (BAL). Total BAL cells were cultured in the presence or absence of lipopolysaccharide. Chemokines were measured in BAL cell supernatants and in cell-free bronchoalveolar lavage fluid (BALF) by enzyme-linked immunoabsorbent assay (ELISA). BAL cell cytospins were stained immunohistochemically for chemokines. BAL cells from asthmatics produced more IL-8 than controls (statistically significant for extrinsic asthma). RANTES was elevated in the BAL cell supernatants of four out of nine intrinsic asthmatics as compared to nonatopic controls (not statistically significant). RANTES levels in the BAL cell supernatants of extrinsic asthmatics were all low. MCP-1 production by BAL cells was similar in all groups. Immunostaining of BAL cell cytospins showed the macrophage to be the predominant positive-staining cell type and correlated well with supernatant data. Measurement of chemokines in BALF showed significantly elevated IL-8 in intrinsic asthma compared to nonatopic controls, but no increase in extrinsic asthmatics relative to atopic control RANTES was elevated in three out of nine BALFs from intrinsic asthmatics compared with nonatopic controls (not statistically significant). MCP-1 was not elevated above control levels in BALF of either asthma group. These results suggest an up-regulation in the production of interleukin-8 and regulated on activation, normal T-cell expressed and secreted, but not monocyte chemotactic protein-1 (MCP-1), by macrophages in the bronchoalveolar lavage of asthmatic subjects. In addition, the data suggest that regulated on activation, normal T-cell, expressed and secreted, may be differentially produced by macrophages in atopic and nonatopic asthma.

Protective responses against skin-dwelling microfilariae of Onchocerca lienalis in severe combined immunodeficient mice.

Inoculation of severe combined immunodeficient (SCID) mice with microfilariae of Onchocerca lienalis results in a sustained infection of the skin, extending for months beyond the point at which the parasites are eliminated from immunocompetent BALB/c controls. Reconstitution of SCID mice with spleen cells, thymocytes, or CD4+-cell-enriched splenocytes from naive BALB/c donors confers the ability to mount a protective immune response, leading to the rapid elimination of microfilariae. High levels of interleukin-5 and low levels of gamma interferon in the sera of reconstituted SCID mice during the destruction of microfilariae suggest that this protective immune response is directed by Th2 lymphocytes, mirroring that observed in immunocompetent controls. Unexpectedly, abbreviation of primary infections of unreconstituted SCID mice with the drug ivermectin induces resistance to reinfection with microfilariae at a level equivalent to that induced in secondarily infected, immunocompetent controls. In contrast to protection mediated by adoptive reconstitution, resistance induced by ivermectin-abbreviated infection occurs in the absence of T cells and in association with negligible levels of serum interleukin-5 and gamma interferon. This points to the activation of some alternative host defense mechanism that operates after the clearance of therapeutic levels of drug. Such a response could have important implications for the treatment of human onchocerciasis and may go some way in explaining the long-term suppression of microfilariae observed in patients after treatment with ivermectin.

The kinetics of immunological responses to microfilariae in a murine host: experimental and mathematical studies.

We present a mathematical model which is used to interpret the dynamics of the immunological response of a mouse host to infection with the filarial worm Onchocerca lienalis. The model mimics changes in worm burden over time post-infection and after reinfection and its behaviour provides a good description of experimental results. Measured production of T-cells and eosinophils is also compared with the predictions of the model. Our results show that the immune response mechanism proposed on the basis of experimental results, involving CD4+ T-cells and eosinophil destruction of the parasite, is supported by the insights gained from the mathematical model. Also, using the parameters estimated to describe the primary infection dynamics, the degree of acquired immunity to secondary infection is also well described by the model. Our analysis highlights the importance of obtaining quantitative measures of the many rate parameters involved in even the simplest interpretations of immunological responses to parasitic infection.

Vaccination generates serum-mediated protection against Onchocerca lienalis microfilariae in the mouse.

Mice vaccinated with an aqueous extract of Onchocerca lienalis microfilariae (mf) plus adjuvant exhibited 51% protection against challenge with live mf. Protection was ablated by prior treatment of the extract with heat or proteinase K, indicating the involvement of proteinaceous epitope(s). A 54% level of protection was conferred on naive mice by passive transfer of serum from vaccinated donors, suggesting that host resistance was principally mediated by humoral components of the immune response. In contrast, the protection induced by sensitization of mice with living mf is transferable with cells, but not serum. Western blot data reveal different antigen recognition profiles for serum antibodies from these two groups of mice. These results indicate that vaccination and infection activate distinct protective mechanisms against Onchocerca mf in the mouse.

Eosinophils are the major effector cells of immunity to microfilariae in a mouse model of onchocerciasis.

Mice inoculated with microfilariae of the filarial nematode Onchocerca lienalis clear their parasites over a period of 3-4 months and are highly resistant to re-infection. We have investigated the comparative roles of the eosinophil, macrophage and neutrophil in effecting this parasite clearance, employing agents specifically to perturb cell function in vivo. Using the anti-IL-5 monoclonal antibody TRFK-5, we show that eosinophils are of primary importance in effecting resistance to re-infection. Ablation of macrophages (with carbon) and neutrophils (with the monoclonal antibody NIMP-R14) had no effect on parasite clearance following re-infection. Neutralization of these 3 cell types during a primary infection showed that while the removal of both eosinophils and macrophages caused a small but significant delay in parasite clearance, the depletion of neutrophils had no effect. This report describes the first direct evidence for eosinophil-mediated killing of microfilariae in vivo, and is consistent with Th-2 cell responses previously described in this model.

Accelerated clearance of Onchocerca microfilariae and resistance to reinfection in interleukin-4 gene knockout mice.

Immunity to Onchocerca microfilariae (mf) in mice is associated with CD4+ Th2 cells and is dependent on IL-5. In view of the role of IL-4 in the development of Th2 cells, we have utilized IL-4 gene knockout mice (IL-4-/-) to investigate microfilarial clearance and resistance to reinfection. Paradoxically, in the absence of IL-4 there is accelerated clearance of microfilariae during a primary infection and unimpeded expression of resistance to reinfection. IL-4-/- mice showed a lack of an IgE response although peripheral eosinophilia was equivalent to wildtype controls. The data presented here suggest that elimination of mf can occur independent of IL-4 and that in this model it may even be detrimental to the host.

Roles for both CD4+ and CD8+ T cells in protective immunity against Onchocerca lienalis microfilariae in the mouse.

Mice inoculated with microfilariae of the filarial nematode Onchocerca lienalis clear their parasites from the skin over a period of 3 to 4 months and are highly resistant to a challenge infection. The adoptive transfer of spleen cells at various time points following primary and secondary infections of mice shows that exposures of 50 days or greater are required for the generation of lymphocytes capable of transferring protection to naive recipients. This adoptive transfer of protection with spleen cells from infection-primed mice partitions with the T lymphocyte population. In contrast, the passive transfer of protection with spleen-derived B cells, or sera taken at various time points following infection was not achieved. Moreover, there was no detectable synergistic effect when B and T cells were co-administered to recipient animals. Depletion of CD4+ and CD8+ T cells with monoclonal antibodies shows that CD8+ T cells have some regulatory effect on parasite establishment early in primary infection, but this is later superseded by CD4+ T cell reactivity that is predominant both when primary infection microfilariae are cleared and also during resistance to reinfection. Measurement of cytokines in the sera of mice undergoing primary and secondary infections support a microfilariae-induced Th2 activity, with high levels of IL-5 that are sustained upon reinfection, and low levels of IFN-gamma that are negligible at the time when mice are most strongly immune.

The effects of H2 and non-H2 genes on the survival of Onchocerca lienalis microfilariae in the mouse.

The effect of H2 and non-H2 genes in a mouse model of protective immunity against Onchocerca lienalis microfilariae have been investigated. Non-H2 effects were determined using CBA, BALB/c, B10, SJL and TO strains. All were permissive for establishment of a primary infection with microfilariae, although significant differences in parasite recoveries were evident amongst the various strains. The effect of H2 genes upon a primary infection was investigated using H2 congenic B10 and BALB strains, B10, B10.S, B10.BR, B10.D2/n, BALB/c, BALB.B, and BALB.K. Significant H2 effects were seen among the relatively weak responder B10 strains, but were not present among the relatively strong responder BALB strains. These results support a dominant effect of non-H2 genes following primary exposure to microfilariae, and a 'fine tuning' effect of H2 genes that is apparent only in weaker responding strains. Upon reinfection of all the strains investigated, a gradation of protection was detected that appeared to be exclusively dependent upon H2.

Protective immunity against Onchocerca volvulus and O. lienalis infective larvae in mice.

Acquired resistance to both Onchocerca volvulus and O. lienalis infective larvae, implanted within micropore chambers, could be induced in mice following immunization with irradiated L3 larvae. In experiments with O. volvulus in BALB/c and BALB/c. By mice, consistent levels of protection (61-75% reductions compared to challenge controls) were achieved with challenge infections of 2 week duration. In DBA/2 mice, levels of protection against O. lienalis were lower and more variable (42-63%): Moreover a 3 week period between challenge and recovery was required before significant reductions in larval recovery became detectable in vaccinated animals. Immunization of CBA or BALB/c mice with O. lienalis microfilariae, or CBA mice with normal or irradiated O. lienalis L3 larvae, failed to induce killing or growth retardation of developing larvae. Preliminary characterization of the effector mechanisms and cytokines associated with protective immunity against O. volvulus infective larvae revealed elevated levels of eosinophils in peripheral blood and within micropore chambers during challenge infections in vaccinated mice. Spleen cells from the same animals stimulated with parasite antigen induced significant levels of IL-5, IL-4 and IFN gamma. These cytokines were barely detectable in antigen stimulated cells from challenge control mice.

Idiotypic vaccination as a treatment for a B cell lymphoma.

To develop a model for the active immunotherapy of human B cell malignancy we vaccinated tumor-bearing animals with a well defined tumor associated Ag, the idiotypic Ig. The tumor used was the mouse B cell lymphoma BCL1, a highly malignant tumor in which transfer of a single tumor cell to a syngeneic mouse is capable of causing disease and eventual death. Varying doses (10(2) to 10(4] of BCL1 cells were given to mice on day 0 of the experiment, and treatment by active immunization was initiated on day 3. Immunization with purified, tumor-derived, idiotypic IgM (BCL1 IgM) coupled to keyhole limpet hemacyanin (KLH) was highly effective in treating mice challenged with 10(2) or 10(3) BCL1 cells, but less effective in mice that had received 10(4) tumor cells. Immunization with unconjugated BCL1 IgM showed no signficant therapeutic benefit. Coupling of the IgM to KLH led to higher levels of anti-idiotypic antibody after immunization; however, the higher levels were probably not responsible for the control of the malignancy as there was no correlation in healthy immunized animals between the levels of anti-idiotypic antibody, measured immediately before tumor challenge, and survival. This lack of correlation is due to the emergence of variant tumors in such protected mice. A more significant factor in the therapeutic advantage of KLH conjugation could be that immunization with BCL1 IgM-KLH led to an earlier induction of the anti-idiotypic response than immunization with BCL1 IgM and, as the BCL1, lymphoma divides rapidly, the speed of induction of the immune response may be important in outstripping tumor cell growth. Mice with BCL1 tumour showed some evidence of immunosuppression as indicated by a reduced ability to mount an immune response against KLH. Although it is not possible to model spontaneous human lymphoma accurately, the generation of a functional anti-idiotypic response capable o eliminating a malignant animal lymphoma in situ opens up the possibility of a limited trial of active immunotherapy in selected human patients.