This single issue from 1973 is important for a number of reasons and that's why - in spite of the fact that it is merely one issue - it finds itself so high on this list. It was the first time the comic books had deliberately tried to avoid the campiness that had become synonymous with The Joker (and indeed Batman) since the debut of the 1960s television series with Adam West and showed the Crown Prince of Crime in a much darker light than he had been shown before. It revolved around The Joker being displeased with the work of five of his henchman, prompting him to try to dispose of them which, in turn, sees the Dark Knight trying to protect a group of people he would normally be fighting against. This was the first time The Joker had displayed such disregard for his own allies. it was also important because The Joker actually got the better of Batman in an exchange but, because he was aware that the Dark Knight was in a weakened state, he refused to finish him because he wouldn't have earned the victory. As such, it was really the first sign that the two characters actually need the challenge of each other's existence to survive.
