Binding properties of anti-histone H1 antibodies suggest that nuclei of necrotic cells rather than cells undergoing apoptosis are engulfed in the LE cell phenomenon

Background

The LE cell phenomenon is a classical test for systemic lupus erythematosus (SLE). Granulocytes engulf bodies, the nature of which has not been unequivocally demonstrated. Hypotheses range from isolated nuclei to intact cells undergoing apoptosis. Using the fact that it is autoantibodies to histone H1 that induce this phenomenon, which thus have to bind the material in question, we investigated histone H1 accessibility to antibodies.

Methods

Polymorphonuclear cells (PMNC) were isolated by centrifugation between Percoll layers, and peripheral blood mononuclear cells over Ficoll Paque. Cells were either used while vital or after the induction of apoptosis (using gliotoxin for PMNC and actinomycin D for peripheral blood mononuclear cells and incubation overnight) or necrosis (heating to 70°C). Apoptosis and necrosis were verified by both Annexin V/propidium iodide and TUNEL on a flow cytometer. Accessibility of histone H1 was likewise tested by fluocytometry, employing a monoclonal anti-histone H1 antibody and a matched control antibody together with a FITC-labelled F(ab')₂ fragment, as well as by SLE sera testing positive for the LE cell phenomenon and healthy control sera.

Results

Apoptosis and necrosis were reliably induced in both PMNC and lymphocytes. The monoclonal anti-histone H1 antibody bound necrotic cells, but did not bind apoptotic or fresh cells. Likewise, LE cell-positive (and thus anti-histone H1-positive) SLE sera bound to necrotic cells, but not to apoptotic or fresh cells, while healthy sera bound to neither cell.

Conclusion

Since, on cells undergoing apoptosis, histone H1 is not accessible to specific antibodies, necrotic cells or isolated nuclei must be engulfed in the LE cell phenomenon.