Opsonization Effects on Mycobacterium avium subsp. paratuberculosis-Macrophage Interactions

Abstract

High antibody titers in ruminants infected with Mycobacterium avium subsp. paratuberculosis correlates with disease progression. Effects of humoral responses during mycobacterial infection are not completely understood. This study suggests that activation status may be an important factor in determining macrophage ability to limit proliferation of opsonized M. avium subsp. paratuberculosis.

Generally, the humoral response present during mycobacterial infections has been considered nonprotective; however, the function and influence of the generated antibodies on the course of infection are incompletely understood. Development of high antibody levels in ruminants infected with Mycobacterium avium subsp. paratuberculosis correlates with deterioration of cell-mediated immunity and development of extensive multibacillary granulomatous lesions (9). The goal of the present study was to test the hypothesis that antibody opsonization of M. avium subsp. paratuberculosis would not enhance mycobactericidal responses in macrophages. We compared the interaction of cultured naive bovine macrophages with M. avium subsp. paratuberculosis opsonized with serum from naive or infected animals with high anti-M. avium subsp. paratuberculosis titers.

Immune serum was pooled from five adult bovine cases of M. avium subsp. paratuberculosis infection that were confirmed by serum enzyme-linked immunosorbent assay for anti-M. avium subsp. paratuberculosis antibodies and gross and microscopic pathology findings. Anti-M. avium subsp. paratuberculosis serum levels were determined for each animal by the Iowa State University Veterinary Diagnostic Laboratory using an IDDEX system according to the manufacturer's instructions. We further titrated the positive serum samples to an endpoint opsonizing activity (indirect immunofluorescence) at >1:500 dilutions for each. Naive serum was pooled from two adult bovines maintained free of infection. Monocytes were generated from peripheral blood mononuclear cells and differentiated into macrophages by 7 days of culture. The 19698 strain of M. avium subsp. paratuberculosis was obtained from the American Type Culture Collection (Manassas, Va.) and conjugated with fluorescein isothiocyanate (FITC) by a previously described method (1). Bacteria were opsonized for 1 h at 37°C with complete or heat-inactivated serum.

We used fluorescence microscopy to determine bacterial uptake/adherence by scoring 100 macrophages cultured on chambered slides (Nalge Nunc International, Naperville, IL) as either containing or lacking FITC-tagged M. avium subsp. paratuberculosis at 1 h postinfection. As shown in Fig. 1, opsonization with naive serum (NS), immune serum (IS), and heat-inactivated immune serum (HIS) led to similar uptake, which was increased over nonopsonized bacteria. Opsonization with heat-inactivated NS (HNS) led to similar uptake as nonopsonized bacteria (P < 0.05). Based on these results, complete IS and NS were similar in the ability to promote macrophage uptake of M. avium subsp. paratuberculosis, as were complement and anti-M. avium subsp. paratuberculosis antibody.

FIG. 1.

Effect of serum opsonization on uptake/adherence of M. avium subsp. paratuberculosis by macrophages. The percentage of macrophages containing at least one bacterium was determined by fluorescence microscopy. Values are the means of three replicates of the experiment ± the standard error of the mean. The asterisks indicate significant decreases in the HNS and no-treatment groups below the IS, HIS, and NS treatment groups (P < 0.05).

To examine kinetics of uptake/adherence, we determined the phagocytic index (mean fluorescent intensity in R1 × percent gated in M1) of macrophages infected with FITC-tagged bacteria by flow cytometry (Fig. 2). At 30 min postinfection, nonopsonized and HIS- and HNS-opsonized bacteria had the lowest and IS- and NS-opsonized bacteria the highest phagocytic indices (P < 0.05). From 60 min on, the phagocytic index of HIS-opsonized bacteria increased to a value similar to IS- and NS-opsonized bacteria, while nonopsonized and HNS-opsonized bacteria remained low (P < 0.05). These data indicate that bacterial uptake over time increased regardless of opsonization treatment. HIS opsonization, however, did not enhance uptake/adherence until 60 min postinfection. Potential mechanisms for this brief delay would include increasing surface expression of Fc γ receptors and/or integrin receptor expression with subsequent potentiation of Fc γ receptors (7, 11).

FIG. 2.

Kinetics of uptake/adherence of opsonized M. avium subsp. paratuberculosis. (A) Dot blot assay demonstrating macrophage gate R1. (B) Histogram showing uninfected macrophages. (C) Histogram demonstrating macrophages containing fluorescently tagged bacteria defined as falling within the M1 region, which was determined by comparison of infected macrophages with uninfected macrophages. (D) Kinetics of adherence/uptake of opsonized and nonopsonized bacteria at 30, 60, and 120 min postinfection by flow cytometry. The phagocytic (Ph.) index was calculated by multiplying the mean fluorescence intensity by the percentage gated in M1. The value reported at each time point is the mean of three replicates of the experiment ± the standard error of the mean. SSC, side scatter; FSC, forward scatter; Gm, geometric mean; CV, coefficient of variance.

We used a standard CFU assay to evaluate proliferation of opsonized and nonopsonized M. avium subsp. paratuberculosis recovered from lysates of infected resting and activated macrophages. Colonies were counted after 4 weeks of incubation in a 37°C incubator. To account for differences in macrophage uptake of opsonized and nonopsonized bacteria, we determined the percent change in CFU between 4 and 48 h postinfection. To prevent antibody-mediated agglutination interference with CFU data, macrophages were washed thoroughly with medium prior to lysis to remove any agglutinating antibody. A significant opsonin effect on M. avium subsp. paratuberculosis growth was not identified in resting macrophages, where variation in CFU recovery was high (data not shown). There was a trend for reduced survival of IS-, HIS-, and NS-opsonized bacteria compared to nonopsonized bacteria in resting cells, again which did not reach statistical significance. CFU variation was reduced in lysates from activated macrophages, which is potentially due to synchronization of macrophage responses following gamma interferon/lipopolysaccharide treatment.

As shown in Fig. 3A, opsonization with IS, which is rich in complement and anti-M. avium subsp. paratuberculosis, appears to favor bacterial growth in activated macrophages. This may be due to predominate complement receptor-mediated uptake, or alternatively interaction between Fc and complement receptors. Use of multiple receptor types for entry of M. tuberculosis into macrophages has been described previously, and it is hypothesized that in vivo this may be the most relevant mechanism of uptake (2, 6a). In contrast, HIS-opsonized bacteria had restricted growth in activated macrophages, comparable to nonopsonized bacteria. This likely was mediated by Fc receptor uptake and initiation of bactericidal mechanisms. Unexpectedly, growth of HNS-opsonized bacteria did not parallel the nonopsonized bacterial data. This is potentially through the influence of additional opsonic factors in the absence of specific antibody and complement, which would include natural antibodies and collectins (5, 8, 10).

FIG. 3.

Growth of opsonized M. avium subsp. paratuberculosis in gamma interferon/lipopolysaccharide-activated macrophages. (A) Growth of M. avium subsp. paratuberculosis determined by CFU assay. Values represent mean numbers of CFU at 48 h divided by the numbers of CFU at 4 h postinfection. Data represent three replicates of the experiment ± the standard error of the mean. The asterisks indicate significant decreases in the no-treatment and HIS treatment groups below the IS and HNS treatment groups (P < 0.05). (B) Dot blot assays demonstrating viable bacteria within the fluorescein-positive gate by flow cytometry. Bacteria were opsonized with immune serum, and complement-mediated uptake into macrophages was blocked by incubation of macrophages with anti-CD18/CD11b. (C) Percent gated mycobacteria within fluorescein-positive gate at 48 h postinfection. Data represents two replicates of the experiment ± the standard deviation. FDA, fluorescein diacetate.

To further examine the ability of Fc receptor-mediated control of bacterial proliferation, we incubated activated macrophages with anti-CD18/CD11b (VMRD, Pullman, WA) to block bovine complement receptor 3. We infected the macrophages with IS-opsonized M. avium subsp. paratuberculosis for 4 h and then washed them to remove extracellular bacteria. As an indicator of viable intracellular bacteria, we measured macrophage lysates at 48 h postinfection for bacterial conversion of nonfluorescent fluorescein diacetate to fluorescein by flow cytometry as previously described (6). As shown in Fig. 3B and C, we observed decreased fluorescence of IS-opsonized bacteria in macrophages where complement receptor 3 was antagonized, indicating lower viability. This supports the observation from the CFU data that antibody opsonization promotes bacterial killing in activated macrophages via Fc receptor-mediated uptake while opsonization with both complement and antibody favors survival.

We next asked whether complement or antibody opsonization would influence intracellular trafficking of M. avium subsp. paratuberculosis. To address this, we examined phagosome acidification by measuring colocalization of FITC-tagged bacteria with Lysotracker Red using laser confocal microscopy as previously described (4). As shown in Table 1, the percent acidified phagosomes containing HIS-opsonized bacteria started high and then decreased over time, while nonopsonized bacteria had the opposite pattern. NS induced a steady level of phagosome acidification. At the last time point, HIS- and NS-opsonized bacteria had lower levels of phagosome acidification than nonopsonized bacteria (P < 0.05). These data demonstrate that opsonins altered phagosome acidification patterns. However, increasing phagosome acidification does not coincide with increased mycobacterial killing, which has also been demonstrated for macrophages infected with M. avium subsp. avium (3). Differences in survival also do not appear to be directly mediated by nitric oxide production, as these values were similar among opsonization groups in activated and resting macrophages (data not shown).

TABLE 1.

Colocalization of Lysotracker Red and M. avium subsp. paratuberculosis as an indicator of bacteria in acidified phagosomes

Treatment	% Colocalizationa
	4 h p.i.d	24 h p.i.	48 h p.i.
None	49.9 (5.5)	51.9 (5.6)	66.9 (5.1)c
NS	43.1 (5.9)b	46.3 (5.4)b	43.6 (5.2)b
HIS	64.9 (5.2)	52.7 (5.6)	45.1 (5.1)bc

^aValues are the means of four replicates of the experiment ± the standard error of the mean.

^bSignificantly different from remaining means within the column (P < 0.05).

^cSignificantly different from remaining means within the row (P < 0.05).

^dp.i., postinfection.

Taken together, these data suggest that macrophage activation status is a factor in determining the ability of macrophages to control proliferation of antibody-opsonized mycobacteria. This conclusion does not support our initial hypothesis that humoral responses would not promote macrophage mycobactericidal functions. It seems, therefore, that given the appropriate environment (successful macrophage activation and predominate antibody opsonization) humoral responses to M. avium subsp. paratuberculosis may have protective capacity.