DEVELOPMENT OF AN ECZEMATOUS SENSITIZATION 35 protein conjugate is formed such as by 2:4 dinitrochlorobenzene. Land- steiner and Jacobs (1) studied compounds of this type and came to the following conclusions: "In the case of a series of substituted benzenes (C1, NO•.) a connection between sensitizing capacity and lability of the C1 or NO2 groups has been shown, indicating the formation of conjugated antigens in the animal." This type of union is, of course, not the ony kind that might develop be- tween the simple chemical and the protein molecule. Gell, et aL (2), dis- cuss the various possibilities: "Many chemical compounds which act as sensitizers have one general feature in common: they are capable of reacting with a group or groups in the molecule of a protein to form a conjugate. A second type of sensitizer is not able itself to combine with protein, but may be metabol- ized in the body to a derivative possessing such powers of combination. There may be a third category of sensitizers which do not react chemi- cally but form strong adsorption complexes with protein. In any case the conjugate or adsorption complex so formed will become antigenically distinct from the parent protein, and if the sensitizer gains access to the tissues of an animal the proteins of the latter may be rendered antigenic." It is important to emphasize the point previously made that the union between the simple chemical and the protein molecule must not too seri- ously derange the structure of the latter. For example, sulfuric acid enters into a rather violent union with protein yet, no one has ever become allergically sensitized to sulfuric acid, and I would doubt that such is bio- logically possible because the reaction between the sulfuric acid and the protein so modifies the protein that it can no longer function as an antigenic entity. These remarks probably pertain equally to all strong acids and alkalies and possibly also apply to a wide variety of substances labeled as "primary irritants." On reflection, it is then seen that the eczematous variety of the delayed allergic sensitization differs from all other allergic sensitizations in at least one fundamental particular, namely, that in this one the full antigen is made in vivo and a constituent of the body partici- pates as a part of the antigenic complex. So far as I know, this is not true for any other form of allergic sensitization. These facts probably have important effects on the subsequent immunologic events and on the nature and location of the pathologic alterations. The exact site at which the protein conjugate is made is not known, but there are reasons to believe that it is ordinarily the epidermis. First, the usual mode of contact with eczematous sensitizers is by application to the skin consequently their method of entry is percutaneous. From a con- sideration of the points previously made as to their chemical reactivity, it is not reasonable to believe that these compounds could get very far into the system without entering into some sort of union with various con-

36 JOURNAL OF THE SOCIETY, OF COSMETIC CHEMISTS stituents. Actually, in the case of mustard gas, in animals that develop a sensitization to it, e.g., the guinea pig and man, Cullumbine (3) has shown that free mustard gas cannot be demonstrated beneath the epidermis. Second, it is known that if the simple chemical is introduced by an extra- epidermal route and precautions are taken to avoid contaminating the epidermis and adjuvants are not employed, an eczematous sensitization de- velops rarely, if at all. Now, if the simple chemical was absorbed as such and conjugates formed elsewhere than the skin, there would be no rational explanation for the foregoing facts. Assuming then a conjugate has been made, what happens to this conju- gate? Before attempting to answer this question, it might be well to digress a moment in order to point out the nature of the problem. If a small area of skin is painted with 2:az dinitrochlorobenzene, upon testing that person approximately three weeks later, assuming they become sensi- tized, all areas of the skin will be found to be reactive to concentrations to which previouslyunexposed individual do not react. The question is, by what /nechanism did the entire integument become allergically hypersensitive? One theory was that the simple chemical when applied to the skin surface either traversed the entire skin in a hypothetical lipid film that envelops the body, or it was disseminated within the epidermis by way of the inter- cellular bridges. (Whether or not these bridges actually function is, so far as I know, not certain.) An alternative hypothesis was that the simple chemical was not distributed in the aforementioned fashion but that it some- how caused antibodies to be manufactured locally, which were then dis- seminated by any route, but which were ultimately brought back to the epidermis and fixed there. None of these hypotheses have stood the test of experimentation in that one can disprove the intra-epidermal transmission by the fact that if an epidermal island is created, i.e., an area of epidermis is surgically isolated from the rest of the epidermis, and the sensitizing ma- terial applied to the island, the entire skin still becomes sensitized. It has also been shown that the site to which the sensitizing simple chemical has been applied can be surgically extirpated any time later than twenty-four hours after the application without interfering in any way with the subse- quent development of the sensitization. This disproves the local manu- facture of antibodies (at least the exclusive local manufacture) and their subsequent dissemination from that site. It would then seem that the conjugate as such must be absorbed at least in part from the site of its formation. The major channel for its absorption is probably the lym- phatics. So far as I know, there is no unequivocal proof for this, but there is much inferential evidence for it which I shall not review. The conjugate is probably then carried to the regional lymph nodes. It is now appropriate to inquire as to the nature of the changes induced by the conjugated protein. If the body regards this conjugated protein as