Anti-GM-CSF autoantibodies in patients with cryptococcal meningitis

Abstract

Cryptococcal meningitis has been described in immunocompromised patients, as well as in those for whom no immune defect has been identified. GM-CSF regulates the function of phagocytes and pulmonary alveolar macrophages, critical elements in cryptococcal control. We performed clinical histories, immunological evaluation, and anticytokine autoantibody screening in four current patients with cryptococcal meningitis and identified and tested 103 archived plasma/cerebrospinal fluid samples from patients with cryptococcal meningitis. We assessed the ability of anti-GM-CSF autoantibody-containing plasmas to inhibit GM-CSF signaling. We recognized anti-GM-CSF autoantibodies in an otherwise healthy female with cryptococcal meningitis who later developed pulmonary alveolar proteinosis (PAP). Her diagnosis prompted screening of patients with cryptococcal meningitis for anticytokine autoantibodies. We identified seven HIV-negative patients with cryptococcal meningitis who tested positive for high-titer anti-GM-CSF autoantibodies. Two of the seven later developed evidence of PAP. Plasma from all patients prevented GM-CSF-induced STAT5 phosphorylation and MIP-1α production in normal PBMCs. This effect was limited to their IgG fraction. Anti-GM-CSF autoantibodies are associated with some cases of cryptococcal meningitis in otherwise immunocompetent patients. These cases need not have associated PAP.

Figure 1

Radiographic and cytopathologic manifestations. A. MRI brain with gadolinium of patient 1 showing two enhancing lesions. B. Later chest CT of patient 1 demonstrating PAP. C. Periodic Acid Schiff (PAS) Diastase stain of BAL fluid showing characteristic findings of PAP with granular proteinaceous material and globules that are PAS positive, diastase resistant. D. Chest CT of patient 3 demonstrating pulmonary cryptococcal lesion with local bony invasion.

Figure 2

Anticytokine autoantibody evaluation. A. Anti-GM-CSF autoantibodies in controls, both diseased (n=60) and healthy (n=43), patients with known acquired pulmonary alveolar proteinosis (PAP), and patients with cryptococcal meningitis (CM) with and without known immunocompromise (IC). B. Multiplex screen for anticytokine autoantibodies against GM-CSF, IFNα, IFNγ, IL-12p70, IL-17A and IL-22 in anti-GM-CSF autoantibody positive (aab+) cryptococcal meningitis patients and 10 normal controls. C. Evaluation of anti-GM-CSF autoantibody positive cryptococcal meningitis patients for other anti-GM-CSF immunoglobulin isotypes and D. anti-GM-CSF IgG subclasses. E. Anti-GM-CSF autoantibodies in CSF.

Figure 3

Inhibitory capacity of anti-GM-CSF autoantibody-containing plasma. A. Normal PBMC were incubated with patient or normal plasma and left unstimulated or stimulated with GM-CSF or IL-3. Intracellular staining for pSTAT-5 was measured by flow cytometry and a stimulation index (ratio of stimulated to unstimulated geometric mean channels) was calculated for each plasma sample tested. B. GM-CSF and IL-3 induction of pSTAT-5 in washed PBMC from 2 patients with anti-GM-CSF autoantibodies. C. Normal PBMC were left unstimulated or stimulated in the presence of either purified IgG or the remaining plasma fraction for all 7 patients. Three of the 7 patients (2 current patients and 1 archived sample) and one normal are shown.

Figure 4

Anti-GM-CSF autoantibody-containing plasma inhibits GM-CSF induced pSTAT-5 in dose-dependent fashion. A. Representative dose-response curves for pSTAT-5 production in normal PBMC incubated with plasma from Patient 3 or normal plasma and stimulated with increasing amounts of GM-CSF. The concentration of GM-CSF required for 50% STAT-5 phosphorylation (EC50) was 798 ng/mL (R²=0.9954) and 0.0371 ng/mL (R²= 0.9999) for Patient 3 and normal plasma, respectively. B. The EC50 was determined from the dose-response curves generated for each of the 7 patient plasmas and 5 normal controls.

Figure 5

Anti-GM-CSF autoantibody-containing plasma inhibits GM-CSF-induced MIP-1α protein expression. Normal PBMC incubated with normal or patient plasma and patient PBMC washed of autologous plasma were left unstimulated or stimulated with GM-CSF and fold-induction of MIP-1α production was determined. For each patient with anti-GM-CSF autoantibodies, values represent an average of 3 independent experiments.