METHODS

Section:

ChooseTop of pageAbstractCASE REPORTSMETHODS <<RESULTSDISCUSSIONReferencesCITING ARTICLES

Bronchoscopy with bronchoalveolar lavage and phlebotomy was performed on healthy volunteers. Bronchoalveolar lavage fluid was processed and alveolar macrophages were obtained as previously described (14). Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient centrifugation. After adherence to plastic for 1 hour, nonadherent cells were removed and lymphocytes were purified by rosetting with sheep red blood cells for 2 hours. Adherent monocytes were harvested by gentle scraping. Alveolar macrophages and peripheral monocytes were plated at 5 × 10⁴ cells per well in 96-well plates. A fungistatic assay was performed as previously described (15). Briefly, 5 × 10³ H. capsulatum yeasts (clinical isolate IU-CT; Indianapolis, IN) in 5% fetal calf serum RPMI with 10 μg/ml gentamicin were placed in each well with monocytes or macrophages, in the presence of anti–TNF-α antibodies (infliximab; Centocor, Inc., Malvern, PA) or a human IgG control (Calbiochem, San Diego, CA) at concentrations ranging from 0.1 to 10 μg/ml. After 24 hours, supernatants were removed, 50 μl [³H] leucine (1.5 μCi; New England Nuclear, Boston, MA; specific activity > 140 Ci/mol) and 5 μl of 10× yeast nitrogen broth were added. Eighteen hours later, 50 μl of l-leucine and 50 μl of sodium hypochlorite were added, and cells were harvested with an automated harvester and counted in a liquid scintillation counter. This assay has been shown to reflect actual proliferation of H. capsulatum and not simply increased uptake of labeled organisms (15). Lymphoproliferation experiments were performed as previously described (16). Briefly, monocytes or macrophages were incubated with or without autologous purified blood lymphocytes, live H. capsulatum, and 1 μg/ml infliximab or control IgG in 20% fetal calf serum RPMI with 10 μg/ml gentamicin for 5 days. Supernatants were harvested and 10 μl of 0.1 mCi/ml of [³H] thymidine (specific activity 6.7 Ci/mol; New England Nuclear) was added. Eighteen hours later, sodium hypochlorite was added and the cells harvested. All supernatants were frozen and used for cytokine measurements. All proliferation experiments were performed in triplicate. Importantly, for these 6-day assays, both infliximab and the control antibody were shown in control experiments to have no effect on cell viability.

TNF-α, IFN-γ, interleukin (IL)-10, and IL-4 measurements were performed using commercially available enzyme-linked immunosorbent assay kits (Quantikine Colorimetric Sandwich ELISA kits; R&D Systems, Minneapolis, MN) following the manufacturer's directions. The lower limit of detection of these kits was 8.0 pg/ml for IFN-γ, 0.12 pg/ml for TNF-α, 0.13 pg/ml for IL-4, and 0.5 pg/ml for IL-10.

RESULTS

Section:

ChooseTop of pageAbstractCASE REPORTSMETHODSRESULTS <<DISCUSSIONReferencesCITING ARTICLES

Proliferation of H. capsulatum in alveolar macrophages and monocytes was determined by the incorporation of [³H] leucine in four normal volunteers in the presence and absence of infliximab or control antibody at various concentrations from 0.1 to 10 μg/ml. Although there is a modest increase in H. capusulatum growth in monocytes and macrophages in the presence of either antibody, there was no difference between the two, suggesting this was a nonspecific antibody response. In Figure 1

Figure 1. Intracellular growth of Histoplasma capsulatum (HC) in alveolar macrophages (AM). AM were exposed to live H. capsulatum in the presence of infliximab or control human IgG antibody as described in METHODS. Growth of HC was measured by incorporation of radioactive leucine 18 hours after infection. HC growth increases modestly after exposure to both antibodies. No difference is noted between the uptake and growth of HC in AMs treated with either infliximab or control IgG antibody (n = 4).

Download Figure | Download Powerpoint

[More] [Minimize]

To assess the effect of TNF-α inhibitors on lymphoproliferative responses to intracellular H. capsulatum, lymphocyte proliferation measured by [³H] thymidine incorporation was determined in the presence and absence of 1.0 μg/ml infliximab. Table 1

We next assessed the production of various cytokines in response to histoplasmosis infection. Table 2

DISCUSSION

Section:

ChooseTop of pageAbstractCASE REPORTSMETHODSRESULTSDISCUSSION <<ReferencesCITING ARTICLES

We report three patients with disseminated histoplasmosis, which occurred while receiving TNF-α inhibitors for rheumatoid arthritis and juvenile rheumatoid arthritis. We further demonstrate in an in vitro model of histoplasmosis that exposure of lymphocytes and alveolar macrophages to H. capsulatum results in T cell proliferation and secretion of IFN-γ and TNF-α, cytokines typical of a TH1 response. Intracellular growth of H. capsulatum in monocytes and alveolar macrophages is enhanced equally in the presence of anti–TNF-α antibodies and control IgG antibodies. However, only antibodies to TNF-α specifically inhibit T cell proliferation. Finally, anti–TNF-α antibodies, but not control antibodies, completely abrogate the H. capsulatum–induced TH1 response in alveolar macrophage cultures and significantly inhibit the response in monocyte cultures. Thus, these data provide evidence that the increased incidence of disseminated histoplasmosis in patients receiving anti–TNF-α therapy results from inhibition of an appropriate TH1 response.

Despite the recognition that TNF-α and IFN-γ are important in host defense against H. capsulatum, previous studies have shown that exogenously administered cytokines do not enhance the fungistatic activity of human monocytes/macrophages (15, 17). Our results demonstrating that anti–TNF-α antibodies were no different than control human IgG in their effect on macrophage fungistatic activity supports this concept. In fact, our observation that infliximab significantly impairs H. capsulatum–induced lymphoproliferation and IFN-γ secretion without affecting macrophage fungistatic activity suggests that TNF-α is more important as a regulator of the cellular immune response than as a direct mediator of macrophage phagocytic function. The role of TNF-α in cellular immune responses is complex. Whereas TNF-α is clearly important in initiating an inflammatory and cellular immune response, recent work suggests that it may also possess anti-inflammatory and immunosuppressive properties in established infections, possibly through inhibition of IL-12 production (18). However, in our model of acute infection, inhibition of TNF-α appears to have a detrimental effect on the initial TH1 response to H. capsulatum.

The lymphoproliferative response to H. capsulatum tended to be greatest when monocytes were used as the antigen presenting cell. Despite this response, the inhibitory effect of infliximab on lymphoproliferation was greatest in alveolar macrophage cultures. This observation supports extensive prior investigations demonstrating that alveolar macrophages are poor accessory cells as compared with blood monocytes (19). It is also intriguing that the H. capsulatum–induced IFN-γ response is more completely inhibited by infliximab in alveolar macrophage cultures than monocyte cultures. These findings suggest that the lung may be uniquely susceptible to the immunosuppressive effects of anti–TNF-α antibodies.

In addition to our 3 patients, 12 cases of histoplasmosis occurring in patients taking TNF-α inhibitors have been reported in the literature (4, 5, 20). Together, infections occurred in 13 patients receiving infliximab and in 2 receiving etanercept. Eleven of the 12 outside cases of histoplasmosis were diagnosed by tissue biopsy. Our three cases were diagnosed noninvasively by antigen detection and serologic methods—antigenuria is present in 92% of patients with disseminated histoplasmosis and can be seen with primary infection after intense exposure. Measurement of Histoplasma antigen in the urine and blood is a rapid, noninvasive technique that can aid in the diagnosis of histoplasmosis in patients with disseminated histoplasmosis (21). We believe that it may also prove useful in the setting of patients receiving TNF-α inhibitors.

On the basis of epidemiologic data and our in vitro work, it would seem prudent to withhold TNF-α inhibitors in patients being treated for histoplasmosis. Chronic antifungal maintenance therapy may be needed if TNF-α inhibiting therapy is resumed. Itraconazole maintenance therapy prevents relapse of histoplasmosis in persons with AIDS (22) and would be expected to be effective in patients who reinstitute TNF-α inhibiting therapy. If chronic maintenance therapy is not implemented, urine antigen screening at 3-month intervals may be performed to look for recurrence.

Screening for exposure to H. capsulatum before beginning anti–TNF-α therapy would also seem reasonable. It is already standard of care to screen for tuberculosis with placement of a PPD before starting therapy with many immunosuppressive agents. However, it is unclear whether remote exposure to H. capsulatum predicts risk of recurrent infection. Modes of acquisition of active infection in immunocompromised hosts may include exogenous exposure or reactivation of latent infection. However, numerous studies have shown that histoplasmosis in immunosuppressed patients is relatively uncommon, even in endemic areas (9, 23, 24). These data suggest that reactivation of “latent” infection during immunosuppression is rare, and exogenous exposure of nonimmune or previously infected hosts is the more common mode of acquisition. Furthermore, screening for histoplasmosis using skin test reactivity or serology has not been shown to predict which immunosuppressed patients will develop active infection (24–26). Thus screening for histoplasmosis in patients about to start immunosuppressive therapy, including anti–TNF-α antibodies, is not recommended at this time.

In summary, patients receiving anti–TNF-α therapy are at risk for developing severe histoplasmosis. This may be due to impairment of the normal TH1 immune response to H. capsulatum. Patients starting anti–TNF-α should be followed closely for the development of granulomatous infections, including histoplasmosis.