LETTERS TO THE EDITOR

The F4/80 monoclonal antibody recognizes a 160-kDa protein expressed on the surface of murine mononuclear phagocytes (1). It has been used extensively in studies of the distribution of tissue macrophages and the role of macrophages in development, autoimmunity, malignancy, viral infection, and the action of macrophage colony-stimulating factor (2-15). A recent publication (16) reports that cosinophils bind the F4/80 antibody and, inter alia, calls into question the interpretation of studies where F4/80 was used as a macrophage marker. The authors extend this observation to propose a link between the differentiation pathways ofeosinophils and macrophages. Such a link is not implausible and I have no reason to resist it as a hypothesis. However, before such leaps of intellect are necessary, the data need to be carefully examined. In the course of developing the immunocytochemical procedures for labeling F4/80 in tissue sections, I became aware very early that F4/80 is an unusual antibody and substantial precautions had to be taken to prevent nonspecific staining. It was for this reason that the standard staining protocols included perfusion fixation with glutaraldehyde and pretreatment of sections with rabbit anti-mouse immunoglobulin (16). Why should F4/80 be any different from any other antibody? During the course oflarge-scale purification of F4/80 antibody, we tried many conventional fractionation procedures and none of them worked. The reason became evident when we resorted to preparative isoelectric focusing, which revealed an isoclectric point of 6.5 (A. I. Cassady and D. A. Hume, unpublished results). Accordingly, unlike most antibodies, F4/80 has a net negative charge at pH 7.4. Whether this fact has any relevance to the binding of F4/80 to eosinophils remains to be seen, but it does indicate to me that an isotype control is not an adequate demonstration of specificity. McGarry and Stewart (15) make the mistaken assertion that in my published studies I might have overlooked staining of eosinophils, because they are normally much less abundant than macrophages. At a later point they claim that 3-7% of normal bone marrow cells arc eosinophils, a proportion similar to the number of F4/80 positive cells reported to be present in adult bone marrow (1). The majority ofthe F4/80 positive cells in sections ofnormal bone marrow arc mononuclear, have a stellate morphology, and form the center of hacmopoietic islands (4). In reproducing immunocytochemisti-y for publication in black and white, it is not possible to do justice to the level of morphological detail discernabic in the perfusion-fixed tissues. The fact is that nuclear morphology is very well defined. In the extensive studies cited above, I have never seen cells with the nuclear morphology of granulocytes expressing F4/80. These studies have included observation oflesions caused in the GI tract by the parasite Nematospiroides dubius and in the liver by Fosciola hepatica, which were mentioned in another context (17). Within the lesions caused by these organisms there were substantial numbers of both cosinophils and macrophages. The cosinophils did not stain with F4/80, whereas adjacent macrophagcs were strongly positive (W. Allan and D. A. Hume, unpublished results). McGarry and Stewart (15) have shown that eosinophils bind the F4/80 antibody weakly, but they have not shown that eosinophils express the F4/80 antigen. They presume that the only way an antibody could be bound to a cell surface is via an Fc receptor. They have not performed quantitative binding assays; even the simple experiment of showing that the labeled antibody could be competed from the cell by unlabeled antibody (i.e., saturability). They have not performed immunoprecipitation to show that the antibody is actually binding to the same molecule on eosinophils. In the absence of such data, the speculation that cosinophils and macrophages have a differentiation pathway is completely