Detection of serum IgG antibodies specific for Wolbachia surface protein in rhesus monkeys infected with Brugia malayi.

The mechanism of lymphedema development in individuals with lymphatic filariasis is presently poorly understood. To investigate whether Wolbachia, symbiotic bacteria living within filarial nematodes, may be involved in disease progression, Wolbachia-specific immune responses were assayed in a group of Brugia malayi-infected rhesus monkeys. Serum IgG antibodies specific for a major Wolbachia surface protein (WSP) were detected in 2 of 12 infected monkeys. It is interesting that both of these monkeys developed lymphedema after becoming amicrofilaremic. WSP-specific antibody responses were temporally associated with increases in antifilarial IgG1 antibodies as well as lymphedema development. These findings suggest that Wolbachia may be important in understanding disease caused by filarial worms.

Interleukin-10 modulates proinflammatory cytokines in the human monocytic cell line THP-1 stimulated with Borrelia burgdorferi lipoproteins.

We determined previously that lipoproteins of Borrelia burgdorferi stimulate inflammatory and anti-inflammatory cytokines (interleukin-10 [IL-10]) in monocytes. IL-10 could have an effect on innate and acquired immune responses to B. burgdorferi and influence the magnitude of the infectious inoculum and disease outcome. To understand the mechanism(s) of IL-10 action during early infection, when innate immunity expressed chiefly by skin macrophages is key, we investigated the effect of exogenous and endogenous IL-10 on the production of the macrophage-derived cytokines IL-6, IL-1beta, IL-12, and tumor necrosis factor alpha (TNF-alpha). We used the THP-1 human monocytic cell line and recombinant lipidated OspA (L-OspA) as the model target cell and stimulant, respectively. To determine the kinetics of cytokine production by THP-1 cells, we stimulated them with L-OspA and also with heat-killed B. burgdorferi cells (HBb) and lipopolysaccharide (LPS). Exogenous IL-10 dampened production of inflammatory cytokines, as elicited by lipoproteins. The inhibition of endogenous IL-10 function by anti-IL-10 antibody reduced the production of IL-12 and IL-6 but not that of IL-1beta and TNF-alpha. An inspection of the kinetics of cytokine production clarified this finding. TNF-alpha was produced prior to, and IL-beta was produced at the same time as, IL-10, whereas IL-6 and IL-12 were produced later. HBb, LPS, and L-OspA yielded similar kinetics of cytokine production. This result reinforces the notion that lipoproteins are the functional molecules in HBb and perhaps in vivo. It indicates also that signaling pathways utilized by LPS and lipoproteins may be extensively shared. The results are consistent with a major role played by IL-10 in controlling the initial phase of infection with this spirochete.

Induction of pro- and anti-inflammatory cytokines by Borrelia burgdorferi lipoproteins in monocytes is mediated by CD14.

We previously showed that heat-killed Borrelia burgdorferi spirochetes and lipidated outer surface protein A (L-OspA) stimulated the in vitro production of interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC) from uninfected humans and rhesus monkeys (G. Giambartolomei et al., Infect. Immun. 66:2691-2697, 1998). Here we demonstrate that uninfected human peripheral blood monocytes, but not B or T cells, are the cells that transcribe the IL-10 cytokine gene in response to heat-killed B. burgdorferi. B. burgdorferi similarly induced an upregulation of the IL-1beta and IL-6 cytokine genes in monocytes and the production of IL-10 and IL-6 in culture supernatants of the human monocytic cell line THP-1. Purified L-OspA (but not unlipidated OspA [U-OspA] or U-OspC) also stimulated the production of both cytokines in THP-1 cells in a dose-dependent fashion, suggesting that acylation of the OspA protein molecule is required for the production of both anti- and pro-inflammatory cytokines in naive monocytes. A lipohexapeptide that contained the tripalmitoyl-modified cysteine motif (Pam3Cys-Hex) of B. burgdorferi lipoproteins but with an arbitrary peptide sequence had the same effect. Monoclonal antibodies (MAbs) MY4 and 60bca, both of which bind to CD14 and are known to block lipopolysaccharide (LPS)-mediated cytokine production, were able to block L-OspA-mediated IL-10 and IL-6 cytokine production. In contrast, MAb 26ic, which also binds to CD14 but does not block LPS function, failed to inhibit L-OspA-mediated cytokine production. These data suggest that activation of monocytes and production of both anti- and pro-inflammatory cytokines induced by lipoproteins proceeds via the CD14 receptor. LPS binding protein was not required for OspA-induced cytokine production. Our results demonstrate that pro- and anti-inflammatory cytokines induced by B. burgdorferi lipoproteins in PBMC are produced by monocytes and that lipoprotein and LPS signaling pathways share at least the initial signaling event that involves the CD14 receptor.

Diminished production of T helper 1 cytokines and lack of induction of IL-2R+ T cells correlate with T-cell unresponsiveness in rhesus monkeys chronically infected with Brugia malayi.

The relationship between antigen-specific responsiveness, parasitic burden, and lymphatic pathology was investigated in nine rhesus monkeys with chronic Brugia malayi infections. Specifically, in vitro proliferation, cytokine gene expression and production, IL-2R expression on T cells, microfilaria (mf) densities, and lymphedema were evaluated. PBMC from three animals (two mf- one mf+) proliferated in response to filarial antigen (responder monkeys, RM) and cells from six animals (5 mf+; one mf-) did not (nonresponder monkeys, NRM). All RM showed lymphedema and none of the NRM did. Antigen-specific IL-2 and IFN-gamma (mRNA and protein) were induced in PBMC from all RM whereas PBMC from only one of six NRM responded with IL-2 and IFN-gamma expression. IL-4 transcripts were induced in PBMC from two of three RM and in cells from all six NRM. IL-10 mRNA expression and protein production were induced in PBMC from two of three RM and in cells from five of six NRM. A marked increase in the frequency of IL-2R+ T cells was observed in antigen-stimulated PBMC cultures of RM but not in those of NRM. The data show that diminished production of Th1 cytokines and lack of induction of IL-2R+T cells may contribute to the unresponsiveness of PBMC from NRM to filarial antigen. They also show that the polarization of immune responses and lymphatic pathology observed in rhesus monkeys is similar to that generally described in human lymphatic filariasis patients.

Histopathological, lymphoscintigraphical, and immunological changes in the inguinal lymph nodes of rhesus monkeys during the early course of infection with Brugia malayi.

The relationship of the early lymphatic pathophysiological alterations with those of tissue inflammatory and cellular responses in the inguinal lymph nodes of Brugia malayi-infected rhesus monkeys was examined. Each of five animals was inoculated subcutaneously in the right calf with 200 third stage larvae (L3) and 5 weeks later, before the onset of patency [10 to 12 weeks postinoculation (PI)], their right inguinal nodes began to show signs of enlargement, becoming most prominent between weeks 10 to 16 PI. Histopathologically, the right nodes had eosinophilic lymphadenitis, lymphoid hyperplasia, and pronounced germinal centers. Lymphoscintigraphy using 99mTc-antimony trisulfide colloid showed pathophysiological alterations of the lymph flow rate in the right leg but not in the left leg at weeks 7 and 15 PI. In vitro blastogenesis to B. malayi antigens at week 10 PI showed the inguinal lymph node cells proliferated more vigorously than did peripheral blood cells early in infection. However, at week 24 PI both lymph node and peripheral blood cells proliferated to antigens. Flow cytometry showed an upregulation of HLA-DR+ lymphocytes in right lymph node cells from infected animals when compared to those from control animals. No changes in CD2, CD4, CD8, CD20, CD29, and CD45R cell numbers in lymph node of infected animals were seen when compared to control animals. Our results show that lymphatic pathology occurs early before the onset of patency, correlating with a marked tissue inflammatory and cellular responses of lymph node cells in B. malayi-infected rhesus monkeys. The rhesus could be an extremely useful model for understanding the evolution of pathology and pathogenesis of the disease.